EP3202900B1 - Polypeptide à dégradation de carbohydrate et ses utilisations - Google Patents
Polypeptide à dégradation de carbohydrate et ses utilisations Download PDFInfo
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- EP3202900B1 EP3202900B1 EP17156056.8A EP17156056A EP3202900B1 EP 3202900 B1 EP3202900 B1 EP 3202900B1 EP 17156056 A EP17156056 A EP 17156056A EP 3202900 B1 EP3202900 B1 EP 3202900B1
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- polypeptide
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- KPTPSLHFVHXOBZ-BIKCPUHGSA-N xylotetraose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)CO[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O[C@H]3[C@@H]([C@@H](O)C(O)OC3)O)OC2)O)OC1 KPTPSLHFVHXOBZ-BIKCPUHGSA-N 0.000 description 1
- ABKNGTPZXRUSOI-UHFFFAOYSA-N xylotriose Natural products OCC(OC1OCC(OC2OCC(O)C(O)C2O)C(O)C1O)C(O)C(O)C=O ABKNGTPZXRUSOI-UHFFFAOYSA-N 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 150000008498 β-D-glucosides Chemical class 0.000 description 1
- 239000002132 β-lactam antibiotic Substances 0.000 description 1
- 229940124586 β-lactam antibiotics Drugs 0.000 description 1
Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
Definitions
- the invention relates to sequences comprising genes that encode polypeptides having lignocellulosic material degrading activity.
- the invention features the full-length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional protein, and variants and fragments of the gene or the amino acid sequence.
- the invention also relates to methods for using these proteins in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins. Also the invention relates to the successful expression of the genes that encode polypeptides having lignocellulosic material degrading activity in a host organism such as Aspergillus niger and/or Rasamsonia emersonii.
- Carbohydrates constitute the most abundant organic compounds on earth. However, much of this carbohydrate is sequestered in complex polymers including starch (the principle storage carbohydrate in seeds and grain), and a collection of carbohydrates and lignin known as lignocellulose.
- starch the principle storage carbohydrate in seeds and grain
- lignocellulose a collection of carbohydrates and lignin known as lignocellulose.
- the main carbohydrate components of lignocellulose are cellulose, hemicellulose, and pectins. These complex polymers are often referred to collectively as lignocellulose.
- Such enzymes may be used to produce sugars for fermentation into chemicals, plastics, such as for instance succinic acid and (bio) fuels, including ethanol, methanol, butanol, synthetic liquid fuels and biogas, for ensiling, and also as enzyme in other industrial processes, for example in the food or feed, textile, pulp or paper or detergent industries and other industries.
- chemicals plastics
- succinic acid and (bio) fuels including ethanol, methanol, butanol, synthetic liquid fuels and biogas, for ensiling
- biogas synthetic liquid fuels and biogas
- WO 2010/113020 describes a polypeptide having alpha-glucuronidase activity and that can degrade glucuronoxylan molecules by hydrolysis of a glycosidic linkage between a MeGlcA residue and a non-terminal xylopyranosyl residue.
- the present invention provides a polypeptide having hemicellulase activity or an activity according to Table 1 which comprises the amino acid sequence set out in SEQ ID NO: 72 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 71, SEQ ID NO: 74, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 75% sequence identity with the sequence set out in SEQ ID NO: 72 or the variant polynucleotide encodes a polypeptide that has at least 75% sequence identity with the sequence set out in SEQ ID NO: 72.
- Temer numbers of the proteins of the invention and their activity Temer number activity activity 1 Temer00088 beta-xylosidase GH3 2 Temer09484 beta-xylosidase GH3 3 Temer08028 alpha-galactosidase GH27 4 Temer02362 alpha-galactosidase GH27 5 Temer08862 alpha-galactosidase GH27 6 Temer04790 xyloglucanase GH12 7 Temer05249 alpha-arabinofuranosidase GH51 8 Temer06848 alpha-arabinofuranosidase GH51 9 Temer02056 alpha-arabinofuranosidase GH51 10 Temer03124 endo-xylanase GH43 11 Temer09491 mannosidase/xylosidase GH31 12 Temer06400 feruloyl esterase CE1 13 Temer08570 endo-xylanase GH
- the polypeptide of the invention has alpha-glucuronidase activity.
- the invention provides a nucleic acid sequence coding for an hemicellulase, whereby the nucleic acid sequence is selected from the group consisting of:
- the invention also provides a nucleic acid construct or vector comprising the polynucleotide of the invention and a cell comprising a polypeptide of the invention or a nucleic acid construct or vector of the invention.
- the cell is a fungal cell, preferably a fungal cell selected from the group consisting of the genera Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Rasamsonia, Thermoascus, Thielavia, Tolypocladium, and Trichoderma.
- a fungal cell preferably a fungal cell selected from the group consisting of the genera Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola
- one or more gene of the cell of the invention is deleted, knocked-out or disrupted in full or in part, wherein optionally the gene encodes for a protease.
- the invention also provides a method for the preparation of a polypeptide according to the invention having hemicellulase or an activity according to Table 1, which method comprises cultivating a cell of the invention under conditions which allow for expression of said polypeptide and, optionally, recovering the expressed polypeptide.
- the invention provides a composition
- a composition comprising: (i) a polypeptide of the invention and; (ii) a cellulase and/or an additional hemicellulase and/or a pectinase, preferably the cellulase is a GH61, cellobiohydrolase, cellobiohydrolase I, cellobiohydrolase II, endo- ⁇ -1,4-glucanase, ⁇ -glucosidase or ⁇ -(1,3)(1,4)-glucanase and/or the hemicellulase is an endoxylanase, ⁇ -xylosidase, ⁇ -L-arabinofuranosidase, ⁇ -D-glucuronidase feruloyl esterase, coumaroyl esterase, ⁇ -galactosidase, ⁇ -galactosidase, ⁇ -mannanase or ⁇ -mannosidase.
- the invention provides a method for the treatment of a substrate comprising hemicellulose, optionally a plant material, which method comprises contacting the substrate with a polypeptide of the invention and/or a composition of the invention.
- Another aspect of the invention relates to the use of a polypeptide of the invention and/or a composition of the invention to produce sugar from a lignocellulosic material.
- the invention also provides:
- polypeptides of the invention having carbohydrate material degrading or carbohydrate hydrolysing activity may be used in industrial processes.
- the invention provides a method for the treatment of a substrate comprising carbohydrate material which method comprises contacting the substrate with a polypeptide or a composition of the invention.
- the invention provides a method for producing a sugar or sugars from lignocellulosic material which method comprises contacting the lignocellulosic material with a polypeptide or a composition of the invention.
- the invention provides a method for producing a fermentation product, which method comprises: producing a fermentable sugar using the described above; and fermenting the resulting fermentable sugar, thereby to produce a fermentation product.
- a polypeptide or a composition of the invention may also be used, for example, in the preparation of a food product, in the preparation of a detergent, in the preparation of an animal feed, in the treatment of pulp or in the manufacture of a paper or in the preparation of a fabric or textile or in the cleaning thereof.
- Table 2 shows codon-pair optimised coding sequence SEQ ID NO's, amino acid sequence SEQ ID NO's, signal sequence SEQ ID NO's, genomic DNA sequence SEQ ID NO's and wild-type coding sequence SEQ ID NO's of the present invention Temer number Codon-pair optimized coding sequence SEQ ID NO: Amino acid sequence SEQ ID NO: Signal sequence SEQ ID NO: Genomic DNA sequence SEQ ID NO: Wild-type coding sequence SEQ ID NO: 1 Temer00088 1 2 3 4 5 2 Temer09484 6 7 8 9 10 3 Temer08028 11 12 13 14 15 4 Temer02362 16 17 18 19 20 5 Temer08862 21 22 23 24 25 6 Temer04790 26 27 28 29 30 7 Temer05249 31 32 33 34 35 8 Temer06848 36 37 38 39 40 9 Temer02056 41 42 43 44 45 10 Temer03124 46 47 48 49 50 11 Temer09491 51 52 53 54 55 12 Temer06400 56 57 58 59 60 13 Temer
- the present invention provides polynucleotides encoding polypeptides, e.g. enzymes which have the ability to modify, for example degrade, a carbohydrate material.
- a carbohydrate material is a material which comprises, consists of or substantially consists of one or more carbohydrates. Enzymes are herein a subclass of polypeptides.
- Substrate also called feedstock
- feedstock is used to refer to a substance that comprises carbohydrate material, which may be treated with enzymes according to the invention, so that the carbohydrate material therein is modified.
- the substrate may contain any other component, including but not limited to non-carbohydrate material and starch.
- the present invention provides polynucleotides encoding polypeptides, e.g. enzymes which have the ability to modify, for example degrade, a carbohydrate material.
- a carbohydrate material is a material which comprises, consists of or substantially consists of one or more carbohydrates. Enzymes are herein a subclass of polypeptides.
- a ⁇ -xylosidase (EC 3.2.1.37) is any polypeptide which is capable of catalyzing the hydrolysis of 1,4- ⁇ -D-xylans, to remove successive D-xylose residues from the non-reducing termini. Such enzymes may also hydrolyze xylobiose. This enzyme may also be referred to as xylan 1,4- ⁇ -xylosidase, 1,4-3-D-xylan xylohydrolase, exo-1,4- ⁇ -xylosidase or xylobiase.
- an ⁇ -galactosidase (EC 3.2.1.22; GH27) is any polypeptide which is capable of catalyzing the hydrolysis of terminal, non-reducing ⁇ -D-galactose residues in ⁇ -D-galactosides, including galactose oligosaccharides, galactomannans, galactans and arabinogalactans. Such a polypeptide may also be capable of hydrolyzing ⁇ -D-fucosides. This enzyme may also be referred to as melibiase.
- a xyloglucanase is a xyloglucan-specific endo- ⁇ -1,4-glucanase, which catalyzes the cleavage of xyloglucan, a backbone of ⁇ 1 ⁇ 4-linked glucose residues, most of which substituted with 1-6 linked xylose side chains.
- an ⁇ -L-arabinofuranosidase (EC 3.2.1.55) is any polypeptide which is capable of acting on ⁇ -L-arabinofuranosides, ⁇ -L-arabinans containing (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans and arabinogalactans.
- This enzyme may also be referred to as ⁇ -N-arabinofuranosidase, arabinofuranosidase or arabinosidase.
- an endoxylanase (EC 3.2.1.8) is any polypeptide which is capable of catalyzing the endo-hydrolysis of 1,4- ⁇ -D-xylosidic linkages in xylans.
- This enzyme may also be referred to as endo-1,4- ⁇ -xylanase or 1,4- ⁇ -D-xylan xylanohydrolase.
- An alternative is EC 3.2.1.136, a glucuronoarabinoxylan endoxylanase, an enzyme that is able to hydrolyze 1,4 xylosidic linkages in glucuronoarabinoxylans.
- a ⁇ -xylosidase (EC 3.2.1.37) is any polypeptide which is capable of catalyzing the hydrolysis of 1,4- ⁇ -D-xylans, to remove successive D-xylose residues from the non-reducing termini. Such enzymes may also hydrolyze xylobiose. This enzyme may also be referred to as xylan 1,4- ⁇ -xylosidase, 1,4- ⁇ -D-xylan xylohydrolase, exo-1,4- ⁇ -xylosidase or xylobiase.
- a ⁇ -mannosidase (EC 3.2.1.25) is any polypeptide which is capable of catalyzing the hydrolysis of terminal, non-reducing ⁇ -D-mannose residues in ⁇ -D-mannosides.
- This enzyme may also be referred to as mannanase or mannase.
- the saccharide may be, for example, an oligosaccharide or a polysaccharide. It may typically catalyze the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar, which is usually arabinose in 'natural' substrates. p-nitrophenol acetate and methyl ferulate are typically poorer substrates.
- This enzyme may also be referred to as cinnamoyl ester hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. It may also be referred to as a hemicellulase accessory enzyme, since it may help xylanases and pectinases to break down plant cell wall hemicellulose and pectin.
- a polypeptide of the invention encodes a polypeptide having at least alpha-glucuronidase activity, tentatively called TEMER07305, having an amino acid sequence according to SEQ ID NO: 72, or a sequence which is a variant thereof, typically functionally equivalent to the polypeptide having the sequence of SEQ ID NO: 72, or a sequence which is a fragment of either thereof.
- This enzyme may also be referred to as alpha-glucuronidase or alpha-glucosiduronase. These enzymes may also hydrolyze 4-O-methylated glucoronic acid, which can also be present as a substituent in xylans.
- Alternative is EC 3.2.1.131: xylan alpha-1,2-glucuronosidase, which catalyses the hydrolysis of alpha-1,2-(4-O-methyl)glucuronosyl links.
- a polypeptide of the invention may have one or more alternative and/or additional carbohydrate degrading and/or carbohydrate hydrolysing activities other than that of alpha-glucuronidase activity, for example one of the other carbohydrate degrading and/or carbohydrate hydrolysing activities mentioned herein.
- Carbohydrate in this context includes all saccharides, for example polysaccharides, oligosaccharides, disaccharides or monosaccharides.
- a polypeptide according to the invention may modify a carbohydrate material by chemically degrading or physically degrading such material or hydrolysing the carbohydrate.
- Chemical modification of the carbohydrate material may result in the degradation of such material, for example by hydrolysis, oxidation or other chemical modification such as by the action of a lyase.
- Physical modification may or may not be accompanied by chemical modification.
- Biomass is a wide-ranging term meaning any source of organic carbon that is renewed rapidly as part of the carbon cycle. Biomass is derived from plant materials but can also include animal materials.
- the composition of lignocellulosic biomass varies, the major component is cellulose (35-50%), followed by xylan (20-35%, a type of hemicellulose) and lignin (10-25%), in addition to minor components such as proteins, oils and ash that make up the remaining fraction of lignocellulosic biomass.
- Lignocellulosic biomass contains a variety of carbohydrates.
- the term carbohydrate is most common in biochemistry, where it is a synonym of saccharide.
- Carbohydrates saccharide are divided into four chemical groupings: monosaccharides, disaccharides, oligosaccharides, and polysaccharides. In general, monosaccharides and disaccharides, which are smaller (lower molecular weight) carbohydrates, are commonly referred to as sugars.
- a non-starch carbohydrate suitable for modification by a polypeptide of the invention is lignocellulose.
- the major polysaccharides comprising different lignocellulosic residues which may be considered as a potential renewable feedstock, are cellulose (glucans), hemicelluloses (xylans, heteroxylans and xyloglucans).
- hemicelluloses xylans, heteroxylans and xyloglucans
- some hemicellulose may be present as glucomannans, for example in wood-derived feedstocks.
- Lignin fills the spaces in the cell wall between cellulose, hemicellulose, and pectin components, especially in xylem tracheids, vessel elements and sclereid cells. It is covalently linked to hemicellulose and, therefore, crosslinks different plant polysaccharides, conferring mechanical strength to the cell wall and by extension the plant as a whole.
- Lignin is a highly hydrophobic crosslinked aromatic polymeric material that is formed by different monolignol monomers, which can be methoxylated to various degrees. There are three monolignol monomers, methoxylated to various degrees: p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol.
- lignols are incorporated into lignin in the form of the phenylpropanoids p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S), respectively.
- Biodegradation of lignin is a prerequisite for processing biofuel from plant raw materials. Lignin can be degraded by applying different pretreatment methods, or by using ligninases or lignin-modifying enzymes (LME's).
- the improving of lignin degradation would drive the output from biofuel processing to better gain or better efficiency factor, for example by improving the accessibility to the (hemi)cellulosic components or by removing lignin-(hemi)cellulose linkages in oligosaccharides released by the action of (hemi)cellulases.
- pectins and other pectic substances such as arabinans may make up considerably proportion of the dry mass of typically cell walls from non-woody plant tissues (about a quarter to half of dry mass may be pectins).
- Cellulose is a linear polysaccharide composed of glucose residues linked by ⁇ -1,4 bonds.
- the linear nature of the cellulose fibers, as well as the stoichiometry of the ⁇ -linked glucose (relative to ⁇ ) generates structures more prone to interstrand hydrogen bonding than the highly branched ⁇ -linked structures of starch.
- cellulose polymers are generally less soluble, and form more tightly bound fibers than the fibers found in starch.
- Hemicellulose is a complex polymer, and its composition often varies widely from organism to organism and from one tissue type to another.
- a main component of hemicellulose is ⁇ -1,4-linked xylose, a five carbon sugar.
- this xylose is often branched at O-3 and/or O-2 and can be substituted with linkages to arabinose, galactose, mannose, glucuronic acid, galacturonic acid or by esterification to acetic acid (and esterification of ferulic acid to arabinose).
- Hemicellulose can also contain glucan, which is a general term for ⁇ -linked six carbon sugars (such as the ⁇ -(1,3)(1,4) glucans and heteroglucans mentioned previously) and additionally glucomannans (in which both glucose and mannose are present in the linear backbone, linked to each other by ⁇ -linkages).
- glucan is a general term for ⁇ -linked six carbon sugars (such as the ⁇ -(1,3)(1,4) glucans and heteroglucans mentioned previously) and additionally glucomannans (in which both glucose and mannose are present in the linear backbone, linked to each other by ⁇ -linkages).
- hemicellulose is very different in dicotyledonous plants (dicots, i.e., plant whose seeds have two cotyledons or seed leaves such as lima beans, peanuts, almonds, peas, kidney beans) as compared to monocotyledonous plants (monocots; i.e., plants having a single cotyledon or seed leaf such as corn, wheat, rice, grasses, barley).
- dicots i.e., plants having a single cotyledon or seed leaf such as corn, wheat, rice, grasses, barley.
- hemicellulose is comprised mainly of xyloglucans that are 1,4- ⁇ -linked glucose chains with 1,6- ⁇ -linked xylosyl side chains.
- heteroxylans In monocots, including most grain crops, the principal components of hemicellulose are heteroxylans. These are primarily comprised of 1,4- ⁇ -linked xylose backbone polymers with 1,3 - ⁇ linkages to arabinose, galactose, mannose and glucuronic acid or 4-O-methyl-glucuronic acid as well as xylose modified by ester-linked acetic acids. Also present are ⁇ glucans comprised of 1,3- and 1,4- ⁇ -linked glucosyl chains. In monocots, cellulose, heteroxylans and ⁇ -glucans may be present in roughly equal amounts, each comprising about 15-25% of the dry matter of cell walls. Also, different plants may comprise different amounts of, and different compositions of, pectic substances. For example, sugar beet contains about 19% pectin and about 21% arabinan on a dry weight basis.
- composition of the invention may be tailored in view of the particular feedstock (also called substrate) which is to be used. That is to say, the spectrum of activities in a composition of the invention may vary depending on the feedstock in question.
- Enzyme combinations or physical treatments can be administered concomitantly or sequentially.
- the enzymes can be produced either exogenously in microorganisms, yeasts, fungi, bacteria or plants, then isolated and added to the lignocellulosic feedstock.
- the enzymes are produced, but not isolated, and crude cell mass fermentation broth, or plant material (such as corn stover), and the like are added to the feedstock.
- the crude cell mass or enzyme production medium or plant material may be treated to prevent further microbial growth (for example, by heating or addition of antimicrobial agents), then added to the feedstock.
- These crude enzyme mixtures may include the organism producing the enzyme.
- the enzyme may be produced in a fermentation that uses feedstock (such as corn stover) to provide nutrition to an organism that produces an enzyme(s).
- feedstock such as corn stover
- plants that produce the enzymes may serve as the lignocellulosic feedstock and be added into lignocellulosic feedstock.
- Endo-1,4- ⁇ -glucanases and exo-cellobiohydrolases (CBH) catalyze the hydrolysis of insoluble cellulose to cellooligosaccharides (cellobiose as a main product), while ⁇ -glucosidases (BGL) convert the oligosaccharides, mainly cellobiose and cellotriose to glucose.
- EG Endo-1,4- ⁇ -glucanases
- CBH exo-cellobiohydrolases
- BGL ⁇ -glucosidases
- Xylanases together with other accessory enzymes, for example ⁇ -L-arabinofuranosidases, feruloyl and acetylxylan esterases, glucuronidases, and ⁇ -xylosidases) catalyze the hydrolysis of part of the hemicelluloses.
- Pectic substances include pectins, arabinans, galactans and arabinogalactans.
- Pectins are the most complex polysaccharides in the plant cell wall. They are built up around a core chain of ⁇ (1,4)-linked D-galacturonic acid units interspersed to some degree with L-rhamnose. In any one cell wall there are a number of structural units that fit this description and it has generally been considered that in a single pectic molecule, the core chains of different structural units are continuous with one another.
- Pectinases include, for example an endo-polygalacturonase, a pectin methyl esterase, an endo-galactanase, a ⁇ -galactosidase, a pectin acetyl esterase, an endo-pectin lyase, pectate lyase, ⁇ -rhamnosidase, an exo-galacturonase, an exo-polygalacturonate lyase, a rhamnogalacturonan hydrolase, a rhamnogalacturonan lyase, a rhamnogalacturonan acetyl esterase, a rhamnogalacturonan galacturonohydrolase, a xylogalacturonase, an ⁇ -arabinofuranosidase.
- galacturonan (homogalacturonan), which may be substituted with methanol on the carboxyl group and acetate on O-2 and O-3; rhamnogalacturonan I (RGI), in which galacturonic acid units alternate with rhamnose units carrying (1,4)-linked galactan and (1,5)-linked arabinan side-chains.
- rhamnogalacturonan I rhamnogalacturonan I
- arabinan side-chains may be attached directly to rhamnose or indirectly through the galactan chains; xylogalacturonan, with single xylosyl units on O-3 of galacturonic acid (closely associated with RGI); and rhamnogalacturonan II (RGII), a particularly complex minor unit containing unusual sugars, for example apiose.
- RGII unit may contain two apiosyl residues which, under suitable ionic conditions, can reversibly form esters with borate.
- a polypeptide of the invention will typically have an activity according to Table 1.
- a polypeptide of the invention may have one or more of the activities set out above in addition to or alternative to that activity.
- a composition of the invention as described herein may have one or more of the activities mentioned above in addition to that provided by a polypeptide of the invention having an activity according to Table 1.
- the invention provides genomic polynucleotide sequences comprising the gene encoding the Temer07305 as well as its coding sequence. Accordingly, the invention relates to an isolated polynucleotide comprising the genomic nucleotide sequence according to the coding nucleotide sequence according to SEQ ID NO: 71 or SEQ ID NO: 74 or SEQ ID NO: 75 and to variants, such as functional equivalents, of either thereof.
- the invention relates to an isolated polynucleotide which is capable of hybridizing selectively, for example under stringent conditions, preferably under highly stringent conditions, with the reverse complement of a polynucleotide comprising the sequence set out in SEQ ID NO: 71 or in SEQ ID NO: 74 or in SEQ ID NO: 75.
- the invention relates to a polynucleotide comprising or consisting essentially of a nucleotide sequence according to SEQ ID NO: 71 or SEQ ID NO: 74 or SEQ ID NO: 75.
- the invention also relates to an isolated polynucleotide comprising or consisting essentially of a sequence which encodes at least one functional domain of a polypeptide according to SEQ ID NO: 72 or a variant thereof, such as a functional equivalent, or a fragment of either thereof.
- gene and “recombinant gene” refer to nucleic acid molecules which may be isolated from chromosomal DNA, which include an open reading frame encoding a protein, e.g. the activity according to the present invention.
- a gene may include coding sequences, non-coding sequences, introns and/or regulatory sequences.
- the term “gene” may refer to an isolated nucleic acid molecule as defined herein.
- a nucleic acid molecule of the present invention such as a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 71 or SEQ ID NO: 74 or SEQ ID NO: 75 or a variant thereof, such as a functional equivalent, can be isolated using standard molecular biology techniques and the sequence information provided herein.
- nucleic acid molecules according to the invention can be isolated using standard hybridization and cloning techniques (e. g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 ).
- nucleic acid molecule encompassing all or a portion of SEQ ID NO: 71 or SEQ ID NO: 74 or SEQ ID NO: 75 may be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence information contained in SEQ ID NO: 71 or in SEQ ID NO: 74 or in SEQ ID NO: 75 .
- PCR polymerase chain reaction
- a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
- the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
- oligonucleotides corresponding to or hybridizable to a nucleotide sequence according to the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
- an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 71 or in SEQ ID NO: 74 or in SEQ ID NO: 75.
- an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is the reverse complement of the nucleotide sequence shown in SEQ ID NO: 71 or in SEQ ID NO: 74 or in SEQ ID NO: 75 or a variant, such as a functional equivalent, of either such nucleotide sequence.
- a nucleic acid molecule which is complementary to another nucleotide sequence is one which is sufficiently complementary to the other nucleotide sequence such that it can hybridize to the other nucleotide sequence thereby forming a stable duplex.
- One aspect of the invention pertains to isolated nucleic acid molecules that encode a polypeptide of the invention or a variant, such as a functional equivalent thereof, for example a biologically active fragment or domain, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.
- a polynucleotide according to the invention may be "isolated".
- an "isolated polynucleotide” or “isolated nucleic acid” is a DNA or RNA that is not immediately contiguous with one or both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
- an isolated nucleic acid includes some or all of the 5' non-coding (e.g. promotor) sequences that are immediately contiguous to the coding sequence.
- the term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an "isolated nucleic acid fragment" is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.
- nucleic acid molecule As used herein, the terms “polynucleotide” or “nucleic acid molecule” are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
- the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
- the nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
- Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a Temer07305 nucleic acid molecule. Also included within the scope of the invention are the complementary strands of the nucleic acid molecules described herein.
- nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule.
- the actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
- a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
- a nucleic acid molecule according to the invention may comprise only a portion or a fragment of the nucleic acid sequence shown in SEQ ID NO: 71 or in SEQ ID NO: 74 or in SEQ ID NO: 75 (or of a variant of either thereof), for example a fragment which can be used as a probe or primer or a fragment encoding a portion of a Temer07305 protein.
- the nucleotide sequence determined from the cloning of the Temer07305 gene and cDNA allows for the generation of probes and primers designed for use in identifying and/or cloning other Temer07305 family members, as well as Temer07305 homologues from other species.
- the probe/primer typically comprises a substantially purified oligonucleotide which typically comprises a region of nucleotide sequence that hybridizes preferably under highly stringent conditions to at least from about 12 to about 15, preferably from about 18 to about 20, preferably from about 22 to about 25, more preferably about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, or about 75 or more consecutive nucleotides of a nucleotide sequence shown in SEQ ID NO: 71 or in SEQ ID NO: 74 or in SEQ ID NO: 75 or of a variant, such as a functional equivalent, of either thereof.
- Probes based on the Temer07305 nucleotide sequences can be used to detect transcripts or genomic Temer07305 sequences encoding the same or homologous proteins for instance in other organisms.
- the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.
- the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.
- Such probes can also be used as part of a diagnostic test kit for identifying cells which express a TEMER09484 protein.
- the polynucleotides herein may be synthetic polynucleotides.
- the synthetic polynucleotides may be optimized in codon use, preferably according to the methods described in WO2006/077258 and/or PCT/EP2007/055943 .
- PCT/EP2007/055943 addresses codon-pair optimization.
- Codon-pair optimization is a method wherein the nucleotide sequences encoding a polypeptide have been modified with respect to their codon-usage, in particular the codon-pairs that are used, to obtain improved expression of the nucleotide sequence encoding the polypeptide and/or improved production of the encoded polypeptide.
- Codon pairs are defined as a set of two subsequent triplets (codons) in a coding sequence.
- nucleic acid construct comprising the polynucleotide as described before.
- Nucleic acid construct is defined herein as a nucleic acid molecule, either single-or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature.
- nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains all the control sequences required for expression of a coding sequence.
- coding sequence as defined herein is a sequence, which is transcribed into mRNA and translated into a transcriptional activator of a protease promoter of the invention.
- a coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
- the nucleic acid has high GC content.
- the GC content herein indicates the number of G and C nucleotides in the construct, divided by the total number of nucleotides, expressed in %.
- the GC content is preferably 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, or in the range of 56-70% or the range of 58-65%.
- the DNA construct comprises a promoter DNA sequence, a coding sequence in operative association with said promoter DNA sequence and control sequences such as:
- translational initiator coding sequence is defined as the nine nucleotides immediately downstream of the initiator or start codon of the open reading frame of a DNA coding sequence.
- the initiator or start codon encodes for the AA methionine.
- the initiator codon is typically ATG, but may also be any functional start codon such as GTG.
- translational termination sequence is defined as the four nucleotides starting from the translational stop codon at the 3' end of the open reading frame or nucleotide coding sequence and oriented in 5' towards 3' direction.
- translational initiator sequence is defined as the ten nucleotides immediately upstream of the initiator or start codon of the open reading frame of a DNA sequence coding for a polypeptide.
- the initiator or start codon encodes for the AA methionine.
- the initiator codon is typically ATG, but may also be any functional start codon such as GTG. It is well known in the art that uracil, U, replaces the deoxynucleotide thymine, T, in RNA.
- Amino acid or nucleotide sequences are said to be homologous when exhibiting a certain level of similarity.
- Two sequences being homologous indicate a common evolutionary origin. Whether two homologous sequences are closely related or more distantly related is indicated by "percent identity” or “percent similarity”, which is high or low respectively. Although disputed, to indicate "percent identity” or “percent similarity”, "level of homology” or “percent homology” are frequently used interchangeably.
- homology in order to determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the complete sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment is carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/based or amino acids. The identity is the percentage of identical matches between the two sequences over the reported aligned region.
- a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
- the skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the homology between two sequences ( Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley ).
- the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. ( Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453 ).
- the algorithm aligns amino acid sequences as well as nucleotide sequences.
- the Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE.
- the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp 276-277, http://emboss.bioinformatics.nl/ ).
- EBLOSUM62 is used for the substitution matrix.
- EDNAFULL is used for nucleotide sequences.
- Other matrices can be specified.
- the optional parameters used for alignment of amino acid sequences are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
- the homology or identity is the percentage of identical matches between the two full sequences over the total aligned region including any gaps or extensions.
- the homology or identity between the two aligned sequences is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment including the gaps.
- the identity defined as herein can be obtained from NEEDLE and is labelled in the output of the program as "IDENTITY".
- the homology or identity between the two aligned sequences is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment.
- the identity defined as herein can be obtained from NEEDLE by using the NOBRIEF option and is labelled in the output of the program as "longest-identity".
- the level of identity (homology) between two sequences is calculated according to the definition of "longest-identity" as can be carried out by using the program NEEDLE.
- the protein sequences of the present invention can further be used as a "query sequence" to perform a search against sequence databases, for example to identify other family members or related sequences. Such searches can be performed using the BLAST programs.
- Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information ( http://www.ncbi.nlm.nih.gov ).
- BLASTP is used for amino acid sequences and BLASTN for nucleotide sequnces.
- the BLAST program uses as defaults:
- the degree of local identity (homology) between the amino acid sequence query or nucleic acid sequence query and the retrieved homologous sequences is determined by the BLAST program. However only those sequence segments are compared that give a match above a certain threshold. Accordingly, the program calculates the identity only for these matching segments. Therefore, the identity calculated in this way is referred to as local identity.
- vectors including cloning and expression vectors, comprising a polynucleotide of the invention encoding a TEMER09484 protein or a functional equivalent thereof and methods of growing, transforming or transfecting such vectors in a suitable host cell, for example under conditions in which expression of a polypeptide of the invention occurs.
- vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
- Polynucleotides of the invention can be incorporated into a recombinant replicable vector, for example a cloning or expression vector.
- the vector may be used to replicate the nucleic acid in a compatible host cell.
- the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
- the vector may be recovered from the host cell. Suitable host cells are described below.
- the vector into which the expression cassette or polynucleotide of the invention is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of the vector will often depend on the host cell into which it is to be introduced.
- a vector according to the invention may be an autonomously replicating vector, i.e. a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e. g. a plasmid.
- the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome (s) into which it has been integrated.
- vector refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
- viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
- Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
- certain vectors are capable of directing the expression of genes to which they are operatively linked.
- expression vectors are referred to herein as "expression vectors".
- expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
- the terms "plasmid” and “vector” can be used interchangeably herein as the plasmid is the most commonly used form of vector.
- the invention is intended to include such other forms of expression vectors, such as cosmid, viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) and phage vectors which serve equivalent functions.
- Vectors according to the invention may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
- a vector of the invention may comprise two or more, for example three, four or five, polynucleotides of the invention, for example for overexpression.
- the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.
- operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell), i.e. the term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
- a regulatory sequence such as a promoter, enhancer or other expression regulation signal "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences or the sequences are arranged so that they function in concert for their intended purpose, for example transcription initiates at a promoter and proceeds through the DNA sequence encoding the polypeptide.
- a vector or expression construct for a given host cell may thus comprise the following elements operably linked to each other in a consecutive order from the 5'-end to 3'-end relative to the coding strand of the sequence encoding the polypeptide of the first invention: (1) a promoter sequence capable of directing transcription of the nucleotide sequence encoding the polypeptide in the given host cell; (2) optionally, a signal sequence capable of directing secretion of the polypeptide from the given host cell into a culture medium; (3) a DNA sequence of the invention encoding a mature and preferably active form of a polypeptide having cellobiohydrolase activity; and preferably also (4) a transcription termination region (terminator) capable of terminating transcription downstream of the nucleotide sequence encoding the polypeptide.
- a promoter sequence capable of directing transcription of the nucleotide sequence encoding the polypeptide in the given host cell
- a signal sequence capable of directing secretion of the polypeptide from the given host cell into a culture
- a 3' untranslated region containing one or more transcription termination sites e.g. a terminator.
- the origin of the terminator is less critical.
- the terminator can, for example, be native to the DNA sequence encoding the polypeptide.
- a yeast terminator is used in yeast host cells and a filamentous fungal terminator is used in filamentous fungal host cells. More preferably, the terminator is endogenous to the host cell (in which the nucleotide sequence encoding the polypeptide is to be expressed).
- a ribosome binding site for translation may be present.
- the coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.
- Enhanced expression of the polynucleotide of the invention may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and/or terminator regions, which may serve to increase expression and, if desired, secretion levels of the protein of interest from the expression host and/or to provide for the inducible control of the expression of a polypeptide of the invention.
- heterologous regulatory regions e.g. promoter, secretion leader and/or terminator regions
- the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
- the vectors, such as expression vectors, of the invention can be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein (e.g. TEMER09484 proteins, mutant forms of TEMER09484 proteins, fragments, variants or functional equivalents thereof.
- the vectors, such as recombinant expression vectors, of the invention can be designed for expression of TEMER09484 proteins in prokaryotic or eukaryotic cells.
- TEMER09484 proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), filamentous fungi, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990 ). Representative examples of appropriate hosts are described hereafter.
- the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
- the vector or expression construct is preferably integrated in the genome of the host cell in order to obtain stable transformants.
- suitable episomal vectors are available into which the expression construct can be incorporated for stable and high level expression, examples thereof include vectors derived from the 2 ⁇ and pKD1 plasmids of Saccharomyces and Kluyveromyces, respectively, or vectors containing an AMA sequence (e.g. AMA1 from Aspergillus).
- the expression constructs are integrated in the host cells genome, the constructs are either integrated at random loci in the genome, or at predetermined target loci using homologous recombination, in which case the target loci preferably comprise a highly expressed gene.
- expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
- vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses
- vectors derived from combinations thereof such as those derived from plasmid and bacteriophage
- control sequences or "regulatory sequences” is defined herein to include at least any component which may be necessary and/or advantageous for the expression of a polypeptide. Any control sequence may be native or foreign to the nucleic acid sequence of the invention encoding a polypeptide. Such control sequences may include, but are not limited to, a promoter, a leader, optimal translation initiation sequences (as described in Kozak, 1991, J. Biol. Chem. 266:19867-19870 ), a secretion signal sequence, a pro-peptide sequence, a polyadenylation sequence, a transcription terminator. At a minimum, the control sequences typically include a promoter, and transcriptional and translational stop signals. As set out above, the term “operably linked” is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the production of a polypeptide.
- control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.
- linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.
- operably linked is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the production of a polypeptide.
- the control sequence may be an appropriate promoter sequence, a nucleic acid sequence, which is recognized by a host cell for expression of the nucleic acid sequence.
- the promoter sequence contains transcriptional control sequences, which mediate the expression of the polypeptide.
- the promoter may be any nucleic acid sequence, which shows transcriptional activity in the cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the cell.
- promoter is defined herein as a DNA sequence that binds RNA polymerase and directs the polymerase to the correct downstream transcriptional start site of a nucleic acid sequence encoding a biological compound to initiate transcription. RNA polymerase effectively catalyzes the assembly of messenger RNA complementary to the appropriate DNA strand of a coding region.
- promoter will also be understood to include the 5'-non-coding region (between promoter and translation start) for translation after transcription into mRNA, cis-acting transcription control elements such as enhancers, and other nucleotide sequences capable of interacting with transcription factors.
- the promoter may be any appropriate promoter sequence suitable for a eukaryotic or prokaryotic host cell, which shows transcriptional activity, including mutant, truncated, and hybrid promoters, and may be obtained from polynucleotides encoding extra-cellular or intracellular polypeptides either homologous (native) or heterologous (foreign) to the cell.
- the promoter may be a constitutive or inducible promoter.
- the promoter is an inducible promoter. More preferably the promoter is a carbohydrate inducible promoter.
- Carbohydrate inducible promoters that are preferably used are selected from a starch-inducible promoter (i.e. a promoter inducible by starch, a monomer, a dimer, a oligomer thereof, such as for example a maltose-inducible promoter, an isomaltose-inducible promoter), a cellulose-inducible promoter (i.e.
- a promoter inducible by pectin, a monomer, a dimer and/or an oligomer thereof such as for example a galacturonic acid-inducible promoter, a rhamnose-inducible promoter), an arabinan-inducible promoter (i.e. a promoter inducible by arabinan, a monomer, a dimer, and/or an oligomer thereof such as for example an arabinose-inducible promoter), a glucose-inducible promoter, a lactose-inducible promoter, a galactose-inducible promoter.
- Other inducible promoters are copper-, oleic acid- inducible promoters.
- Promoters suitable in filamentous fungi are promoters which may be selected from the group, which includes but is not limited to promoters obtained from the polynucleotides encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus gpdA promoter, A. niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger or A. awamori glucoamylase (glaA), A. niger or A. awamori endoxylanase ( xlnA) or beta-xylosidase ( xln D), T.
- promoters which may be selected from the group, which includes but is not limited to promoters obtained from the polynucleotides encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus gpd
- reesei cellobiohydrolase I CBHI
- R. miehei lipase R. oryzae alkaline protease
- A. oryzae triose phosphate isomerase A. nidulans acetamidase, Fusarium venenatum amyloglucosidase ( WO 00/56900 ), Fusarium venenatum Dania ( WO 00/56900 ), Fusarium venenatum Quinn ( WO 00/56900 ), Fusarium oxysporum trypsin-like protease ( WO 96/00787 ), Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endo
- niger neutral alpha-amylase and A. oryzae triose phosphate isomerase mutant, truncated, and hybrid promoters thereof.
- Other examples of promoters are the promoters described in WO2006/092396 and WO2005/100573 .
- An even other example of the use of promoters is described in WO2008/098933 .
- Preferred carbohydrate inducible promoters which can be used in filamentous fungi are the A. oryzae TAKA amylase, A. niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger or A. awamori glucoamylase (glaA), A. niger or A.
- Trichoderma reesei beta-glucosidase Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a hybrid of the promoters from the polyn
- Activators are also sequence-specific DNA binding proteins that induce promoter activity.
- promoters from Gram-positive microorganisms include, but are not limited to, two-component systems (PhoP-PhoR, DegU-DegS, Spo0A-Phosphorelay), LevR, Mry and GltC.
- Production of secondary sigma factors can be primarily responsible for the transcription from specific promoters.
- Examples from Gram-positive microorganisms include, but are not limited to, the promoters activated by sporulation specific sigma factors: ⁇ F, ⁇ E, ⁇ G and ⁇ K and general stress sigma factor, ⁇ B.
- Attenuation and antitermination also regulates transcription. Examples from Gram-positive microorganisms include, but are not limited to, trp operon and sacB gene.
- Other regulated promoters in expression vectors are based the sacR regulatory system conferring sucrose inducibility ( Klier AF, Rapoport G. Annu Rev Microbiol. 1988; 42:65-95 ).
- Suitable inducible promoters useful in bacteria include: promoters from Gram-positive microorganisms such as, but are not limited to, SP01-26, SP01-15, veg, pyc (pyruvate carboxylase promoter), and amyE.
- promoters from Gram-negative microorganisms include, but are not limited to, tac, tet, trp-tet, Ipp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, ⁇ -PR, and ⁇ -PL.
- promoters useful in bacterial cells include the ⁇ -amylase and SPo2 promoters as well as promoters from extracellular protease genes.
- a suitable promoter is the promoter obtained from the E. coli lac operon.
- Another example is the promoter of the Streptomyces coelicolor agarase gene (dagA).
- dagA Streptomyces coelicolor agarase gene
- Another example is the promoter of the Bacillus lentus alkaline protease gene (aprH).
- aprH Bacillus lentus alkaline protease gene
- sacB Bacillus subtilis levansucrase gene
- Another example is the promoter of the Bacillus subtilis alphaamylase gene (amyF).
- Another example is the promoter of the Bacillus licheniformis alphaamylase gene (amyL).
- Another example is the promoter of the Bacillus stearothermophilus maltogenic amylase gene (amyM).
- Another example is the promoter of the Bacillus amyloliquefaciens alpha-amylase gene (amyQ).
- Another example is a "consensus” promoter having the sequence TTGACA for the "-35" region and TATAAT for the "-10" region.
- Another example is the promoter of the Bacillus licheniformis penicillinase gene (penP).
- Another example are the promoters of the Bacillus subtilis xylA and xylB genes.
- the promoter sequence is from a highly expressed gene.
- preferred highly expressed genes from which promoters may be selected and/or which are comprised in preferred predetermined target loci for integration of expression constructs include but are not limited to genes encoding glycolytic enzymes such as triose-phosphate isomerases (TPI),glyceraldehyde-phosphate dehydrogenases (GAPDH), phosphoglycerate kinases (PGK), pyruvate kinases (PYK or PKI), alcohol dehydrogenases (ADH), as well as genes encoding amylases, glucoamylases, proteases, xylanases, cellobiohydrolases, ⁇ -galactosidases, alcohol (methanol) oxidases, elongation factors and ribosomal proteins.
- TPI triose-phosphate isomerases
- GPDH glycolytic enzymes
- PGK phosphoglycerate kinases
- suitable highly expressed genes include e. g. the LAC4 gene from Kluyveromyces sp., the methanol oxidase genes (AOX and MOX) from Hansenula and Pichia, respectively, the glucoamylase (glaA) genes from A. niger and A. awamori, the A. oryzae TAKA-amylase gene, the A. nidulans gpdA gene and the T. reesei cellobiohydrolase genes.
- LAC4 gene from Kluyveromyces sp.
- AOX and MOX methanol oxidase genes
- glaA glucoamylase
- Promoters which can be used in yeast include e.g. promoters from glycolytic genes, such as the phosphofructokinase (PFK), triose phosphate isomerase (TPI), glyceraldehyde-3 -phosphate dehydrogenase (GPD, TDH3 or GAPDH), pyruvate kinase (PYK), phosphoglycerate kinase (PGK) promoters from yeasts or filamentous fungi; more details about such promoters from yeast may be found in ( WO 93/03159 ).
- PFK phosphofructokinase
- TPI triose phosphate isomerase
- GPD glyceraldehyde-3 -phosphate dehydrogenase
- PYK pyruvate kinase
- PGK phosphoglycerate kinase
- promoters are ribosomal protein encoding gene promoters, the lactase gene promoter (LAC4), alcohol dehydrogenase promoters (ADHI, ADH4, and the like), and the enolase promoter (ENO).
- LAC4 lactase gene promoter
- ADHI, ADH4, and the like alcohol dehydrogenase promoters
- ENO enolase promoter
- Other promoters, both constitutive and inducible, and enhancers or upstream activating sequences will be known to those of skill in the art.
- the promoters used in the host cells of the invention may be modified, if desired, to affect their control characteristics. Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters, which are well known to the person skilled in the art.
- Suitable promoters in eukaryotic host cells may be GAL7, GAL10, or GAL1, CYC1, HIS3, ADH1, PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO1, TPI1, and AOX1.
- Other suitable promoters include PDC1, GPD1, PGK1, TEF1, and TDH3.
- Examples of carbohydrate inducible promoters which can be used are GAL promoters, such as GAL1 or GAL10 promoters.
- Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
- enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
- the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a filamentous fungal cell to terminate transcription.
- the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator, which is functional in the cell, may be used in the present invention.
- the control sequence may also be a terminator.
- Preferred terminators for filamentous fungal cells are obtained from the genes encoding A. oryzae TAKA amylase, A. niger glucoamylase (glaA), A. nidulans anthranilate synthase, A. niger alpha-glucosidase, trpC gene and Fusarium oxysporum trypsin-like protease.
- the control sequence may also include a suitable leader sequence, a non-translated region of a mRNA which is important for translation by the filamentous fungal cell.
- the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence, which is functional in the cell, may be used in the present invention.
- Preferred leaders for filamentous fungal cells are obtained from the genes encoding A. oryzae TAKA amylase and A. nidulans triose phosphate isomerase and A. niger glaA. Other preferred sequences are isolated and/or disclosed in WO2006/077258 .
- control sequences may be isolated from the Penicillium IPNS gene, or pcbC gene, the beta tubulin gene. All the control sequences are cited in WO 01/21779 .
- the control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the filamentous fungal cell as a signal to add polyadenosine residues to transcribed mRNA.
- Any polyadenylation sequence, which is functional in the cell, may be used in the present invention.
- Preferred polyadenylation sequences for filamentous fungal cells are obtained from the genes encoding A. oryzae TAKA amylase, A. niger glucoamylase, A. nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease and A. niger alpha-glucosidase.
- an appropriate signal sequence can be added to the polypeptide in order to direct the de novo synthesized polypeptide to the secretion route of the host cell.
- the person skilled in the art knows to select an appropriate signal sequence for a specific host.
- the signal sequence may be native to the host cell, or may be foreign to the host cell.
- a signal sequence from a protein native to the host cell can be used.
- said native protein is a highly secreted protein, i.e. a protein that is secreted in amounts higher than 10% of the total amount of protein being secreted.
- the signal sequences preferably used according to the invention are for example: pmeA.
- the polypeptide of the invention can be fused to a secreted carrier protein, or part thereof.
- a secreted carrier protein or part thereof.
- Such chimeric construct is directed to the secretion route by means of the signal sequence of the carrier protein, or part thereof.
- the carrier protein will provide a stabilizing effect to the polypeptide according to the invention and or may enhance solubility.
- Such carrier protein may be any protein.
- a highly secreted protein is used as a carrier protein.
- the carrier protein may be native or foreign to the polypeptide according to the invention.
- the carrier protein may be native of may be foreign to the host cell.
- carrier proteins examples include glucoamylase, prepro sequence of alpha-Mating factor, cellulose binding domain of Clostridium cellulovorans cellulose binding protein A, glutathione S-transferase, chitin binding domain of Bacillus circulans chitinase A1, maltose binding domain encoded by the malE gene of E. coli K12, beta-galactosidase, and alkaline phosphatase.
- a preferred carrier protein for expression of such chimeric construct in Aspergillus cells is glucoamylase.
- the carrier protein and polypeptide according to the invention may contain a specific amino acid motif to facilitate isolation of the polypeptide; the polypeptide according to the invention may be released by a special releasing agent.
- the releasing agent may be a proteolytic enzyme or a chemical agent.
- An example of such amino acid motif is the KEX protease cleavage site, which is well-known to the person skilled in the art.
- a signal sequence can be used to facilitate secretion and isolation of a protein or polypeptide of the invention.
- Signal sequences are typically characterized by a core of hydrophobic amino acids, which are generally cleaved from the mature protein during secretion in one or more cleavage events.
- Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway.
- the signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved.
- the protein can then be readily purified from the extracellular medium by known methods.
- the signal sequence can be linked to the protein of interest using a sequence, which facilitates purification, such as with a GST domain.
- the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide, which facilitates purification of the fused polypeptide.
- the marker sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci.
- hexa-histidine provides for convenient purification of the fusion protein.
- the HA tag is another peptide useful for purification which corresponds to an epitope derived of influenza hemaglutinin protein, which has been described by Wilson et al., Cell 37:767 (1984 ), for instance.
- a TEMER09484 fusion protein of the invention is produced by standard recombinant DNA techniques.
- DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
- the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
- PCR amplification of gene fragments can be carried out using anchor primers, which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992 ).
- anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
- many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
- a TEMER09484 -encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the TEMER09484 protein.
- the polynucleotides of the present invention as described herein may be over-expressed in a microbial strain of the invention compared to the parent microbial strain in which said gene is not over-expressed.
- Over-expression of a polynucleotide sequence is defined herein as the expression of the said sequence gene which results in an activity of the enzyme encoded by the said sequence in a microbial strain being at least about 1.5-fold the activity of the enzyme in the parent microbial; preferably the activity of said enzyme is at least about 2-fold, more preferably at least about 3-fold, more preferably at least about 4-fold, more preferably at least about 5-fold, even more preferably at least about 10-fold and most preferably at least about 20-fold the activity of the enzyme in the parent microbial.
- the vector may further include sequences flanking the polynucleotide giving rise to RNA which comprise sequences homologous to eukaryotic genomic sequences or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of a host cell.
- An integrative cloning vector may integrate at random or at a predetermined target locus in the chromosome(s) of the host cell into which it is to be integrated.
- an integrative cloning vector may comprise a DNA fragment which is homologous to a DNA sequence in a predetermined target locus in the genome of host cell for targeting the integration of the cloning vector to this predetermined locus.
- the cloning vector may be preferably linearized prior to transformation of the host cell. Linearization may preferably be performed such that at least one but preferably either end of the cloning vector is flanked by sequences homologous to the target locus.
- the length of the homologous sequences flanking the target locus is preferably at least about 0.1kb, such as about at least 0.2kb, more preferably at least about 0.5 kb, even more preferably at least about 1 kb, most preferably at least about 2 kb.
- the parent host strains may be modified for improved frequency of targeted DNA integration as described in WO05/095624 and/or WO2007/115886 .
- the deletion example provided in the present invention uses the promoter of the gene as 5'-flank and the gene as the 3'-flank to insert a selection marker between the promoter and gene, thereby disturbing (i.e. functionally inactivating) gene transcription.
- the gene sequences given above can be used to make similar functionally inactivated genes.
- the genes may be split in two, yielding a 5'-flank and a 3'-flank, but the gene may also be used to clone a larger piece of genomic DNA containing the promoter and terminator regions of the gene, which than can function as 5'-flank and a 3'-flanks.
- the vector system may be a single vector, such as a single plasmid, or two or more vectors, such as two or more plasmids, which together contain the total DNA to be introduced into the genome of the host cell.
- the vector may contain a polynucleotide of the invention oriented in an antisense direction to provide for the production of antisense RNA.
- Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
- transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic lipid-mediated transfection or electroporation.
- Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd,ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 ), Davis et al., Basic Methods in Molecular Biology (1986 ) and other laboratory manuals.
- the person skilled in the art knows how to transform cells with the one or more expression cassettes and the selectable marker.
- the skilled person may use one or more expression vectors, wherein the one or more cloning vectors comprise the expression cassettes and the selectable marker.
- Transformation of the mutant microbial host cell may be conducted by any suitable known methods, including e.g. electroporation methods, particle bombardment or microprojectile bombardment, protoplast methods and Agrobacterium mediated transformation (AMT).
- electroporation methods particle bombardment or microprojectile bombardment
- protoplast methods Preferably the protoplast method is used.
- Procedures for transformation are described by J.R.S. Fincham, Transformation in fungi. 1989, Microbiological reviews. 53, 148-170 .
- Transformation may involve a process consisting of protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se.
- Suitable procedures for transformation of Aspergillus cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81:1470-1474 .
- Suitable procedures for transformation of Aspergillus and other filamentous fungal host cells using Agrobacterium tumefaciens are described in e.g. De Groot et al., Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol. 1998, 16:839-842 . Erratum in: Nat Biotechnol 1998 16:1074 .
- a suitable method of transforming Fusarium species is described by Malardier et al., 1989, Gene 78:147156 or in WO 96/00787 .
- Other methods can be applied such as a method using biolistic transformation as described in: Christiansen et al., Biolistic transformation of the obligate plant pathogenic fungus, Erysiphe graminis f.sp. hordei. 1995, Curr Genet. 29:100-102 .
- Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M.
- an expression vector may comprise multiple expression cassettes to increase the amount of copies of the polynucleotide(s) to be transformed.
- Another way could be to choose different control sequences for the different polynucleotides, which - depending on the choice - may cause a higher or a lower production of the desired polypeptide(s).
- the cells transformed with the selectable marker can be selected based on the presence of the selectable marker.
- the selectable marker usually when the cell is transformed with all nucleic acid material at the same time, when the selectable marker is present also the polynucleotide(s) encoding the desired polypeptide(s) are present.
- selectable markers include, but are not limited to, those which confer resistance to drugs or which complement a defect in the host cell. They include e. g. versatile marker genes that can be used for transformation of most filamentous fungi and yeasts such as acetamidase genes or cDNAs (the amdS, niaD, facA genes or cDNAs from A.
- nidulans A. oryzae or A. niger
- genes providing resistance to antibiotics like G418, hygromycin, bleomycin, kanamycin, methotrexate, phleomycin orbenomyl resistance (benA).
- specific selection markers can be used such as auxotrophic markers which require corresponding mutant host strains: e.g. URA3 (from S. cerevisiae or analogous genes from other yeasts), pyrG or pyrA (from A. nidulans or A. niger), argB (from A. nidulans or A. niger) or trpC.
- the selection marker is deleted from the transformed host cell after introduction of the expression construct so as to obtain transformed host cells capable of producing the polypeptide which are free of selection marker genes.
- markers include ATP synthetase, subunit 9 (oliC), orotidine-5'-phosphate decarboxylase (pvrA), the bacterial G418 resistance gene (this may also be used in yeast, but not in fungi), the ampicillin resistance gene (E. coli), the neomycin resistance gene (Bacillus) and the E. coli uidA gene, coding for ⁇ -glucuronidase (GUS).
- Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
- Fusion vectors add a number of amino acids to a protein encoded therein, e.g. to the amino terminus of the recombinant protein.
- Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
- a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
- the expression vectors will preferably contain selectable markers.
- markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracyline or ampicillin resistance for culturing in E. coli and other bacteria.
- Vectors preferred for use in bacteria are for example disclosed in WO-A1-2004/074468 , which are hereby enclosed by reference. Other suitable vectors will be readily apparent to the skilled artisan.
- secretion signal may be incorporated into the expressed polypeptide.
- the signals may be endogenous to the polypeptide or they may be heterologous signals.
- the TEMER09484 polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions.
- a region of additional amino acids, particularly charged amino acids may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
- peptide moieties may be added to the polypeptide to facilitate purification
- the invention provides an isolated polypeptide having the amino acid sequence according to SEQ ID NO: 72, and an amino acid sequence obtainable by expressing the polynucleotide of SEQ ID NO: 71 or SEQ ID NO: 74 or SEQ ID NO: 75 in an appropriate host. Also, a peptide or polypeptide comprising a variant of the above polypeptides, such as a functional equivalent, is comprised within the present invention.
- the above polypeptides are collectively comprised in the term "polypeptides according to the invention"
- variant peptide or “variant polypeptide” is defined herein as a peptide or polypeptide, respectively, comprising one or more alterations, such as substitutions, insertions, deletions and/or truncations of one or more specific amino acid residues at one or more specific positions in the peptide or polypeptide, respectively.
- a variant signal peptide is a signal peptide comprising one or more alterations, such as substitutions, insertions, deletions and/or truncations of one or more specific amino acid residues at one or more specific positions in the signal peptide.
- polynucleotide is identical to the term “nucleic acid molecule” and can herein be read interchangeably.
- the term refers to a polynucleotide molecule, which is a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule, either single stranded or double stranded.
- RNA ribonucleic acid
- DNA deoxyribonucleic acid
- a polynucleotide may either be present in isolated form, or be comprised in recombinant nucleic acid molecules or vectors, or be comprised in a host cell.
- variant polynucleotide is defined herein as a polynucleotide comprising one or more alterations, such as substitutions, insertions, deletions and/or truncations of one or more nucleotides at one or more specific positions in the polynucleotide.
- peptide and oligopeptide are considered synonymous (as is commonly recognized) and each term can be used interchangeably, as the context requires, to indicate a chain of at least two amino acids coupled by peptidyl linkages.
- polypeptide is used herein for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. The one-letter code of amino acids used herein is commonly known in the art and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd,ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 )
- isolated polypeptide or protein is intended a polypeptide or protein removed from its native environment.
- recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988 ).
- the Temer07305 protein according to the invention can be recovered and purified from recombinant cell cultures by methods known in the art. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
- HPLC high performance liquid chromatography
- Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
- the invention also features biologically active fragments of the polypeptides according to the invention.
- Biologically active fragments of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the Temer07305 protein (e.g., the amino acid sequence of SEQ ID NO: 72), which include fewer amino acids than the full length protein but which exhibit at least one biological activity of the corresponding full-length protein.
- biologically active fragments comprise a domain or motif with at least one activity of the Temer07305 protein.
- a biologically active fragment of a protein of the invention can be a polypeptide which is, for example, about 10, about 25, about 50, about 100 or more amino acids in length or at least about 100 amino acids, at least 150, 200, 250, 300, 350, 400 amino acids in length, or of a length up the total number of amino acids of polypeptide of the invention.
- biologically active portions in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the biological activities of the native form of a polypeptide of the invention.
- the invention also features nucleic acid fragments which encode the above biologically active fragments of the Temer07305 protein.
- Improved Temer07305 proteins are proteins wherein at least one biological activity is improved. Such proteins may be obtained by randomly introducing mutations along all or part of the Temer07305 coding sequence, such as by saturation mutagenesis, and the resulting mutants can be expressed recombinantly and screened for biological activity. For instance, the art provides for standard assays for measuring the enzymatic activity of the protein of the invention and thus improved proteins may easily be selected.
- Variants of the genes of the present invention leading to an increased level of mRNA and/or protein, resulting in more an activity according to Table 1 may be obtained by the polynucleotide sequences of said genes. Among such modifications are included:
- Preferred methods to isolate variants with improved catalytic properties or increased levels of mRNA or protein are described in WO03/010183 and WO03/01311 .
- Preferred methods to optimize the codon usage in parent microbial strains are described in PCT/EP2007/05594 .
- Preferred methods for the addition of stabilizing elements to the genes encoding the cellobiohydrolase of the invention are described in WO2005/059149 .
- the protein of the invention has an amino acid sequence according to SEQ ID NO: 72.
- the polypeptide of the invention is substantially homologous to the amino acid sequence according to SEQ ID NO: 72 and retains at least one biological activity of a polypeptide according to SEQ ID NO: 72, yet differs in amino acid sequence due to natural variation or mutagenesis as described.
- the protein of the invention has an amino acid sequence encoded by an isolated nucleic acid fragment capable of hybridizing to a nucleic acid according to SEQ ID NO: 71 or SEQ ID NO: 74 or SEQ ID NO: 75, preferably under highly stringent hybridization conditions.
- the Temer07305 protein or the protein of the invention is preferably a protein which comprises an amino acid sequence at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 95%, 96%, 97%, 98%, 97%, 98%, 99%, 99.8%, 99.9% or more homologous to the amino acid sequence shown in SEQ ID NO: 72 and, typically, retains at least one functional activity of the polypeptide according to SEQ ID NO: 72.
- the polypeptide of the invention may comprise the amino acid sequence set out in SEQ ID NO: 72 or an amino acid sequence that differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids from the amino acid sequence set out in SEQ ID NO: 72 and whereby the polypeptide still has the activity or function of the polypeptide of the invention.
- these minor amino acid changes in the polypeptide of the invention may be present (for example naturally occurring mutations) or made (for example using r-DNA technology) without loss of the protein function or activity.
- a property of the polypeptide may change (for example its thermostability) but the polypeptide may keep its hemicellulase activity.
- a mutation is present which is not close to the active site, binding domain, or other functional domain, less effect may be expected.
- a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level.
- a variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display).
- libraries of fragments of the coding sequence of a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening a subsequent selection of variants.
- a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector.
- an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.
- Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention ( Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815 ; Delgrave et al. (1993) Protein Engineering 6(3): 327-331 ).
- Fragments of a polynucleotide according to the invention may also comprise polynucleotides not encoding functional polypeptides. Such polynucleotides may function as probes or primers for a PCR reaction.
- Nucleic acids according to the invention irrespective of whether they encode functional or non-functional polypeptides can be used as hybridization probes or polymerase chain reaction (PCR) primers.
- Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having a Temer07305 activity include, inter alia, (1) isolating the gene encoding the Temer07305 protein, or allelic variants thereof from a cDNA library e.g. from suitable microorganisms; (2) in situ hybridization (e.g.
- FISH FISH to metaphase chromosomal spreads to provide precise chromosomal location of the Temer07305 gene as described in Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988 ); (3) Northern blot analysis for detecting expression of Temer07305 mRNA in specific tissues and/or cells and 4) probes and primers that can be used as a diagnostic tool to analyse the presence of a nucleic acid hybridizable to the Temer07305 probe in a given biological (e.g. tissue) sample.
- a nucleic acid hybridizable to the Temer07305 probe in a given biological (e.g. tissue) sample.
- a method of obtaining a functional equivalent of a Temer07305 gene entails obtaining a labelled probe that includes an isolated nucleic acid which encodes all or a portion of the protein sequence according to SEQ ID NO: 72 or a variant thereof; screening a nucleic acid fragment library with the labelled probe under conditions that allow hybridization of the probe to nucleic acid fragments in the library, thereby forming nucleic acid duplexes, and preparing a full-length gene sequence from the nucleic acid fragments in any labelled duplex to obtain a gene related to the Temer07305 gene.
- a Temer07305 nucleic acid of the invention is at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to a nucleic acid sequence shown in SEQ ID NO: 71 or in SEQ ID NO: 74 or in SEQ ID NO: 75 or the complement thereof.
- host cells comprising a polynucleotide or vector of the invention.
- the polynucleotide may be heterologous to the genome of the host cell.
- heterologous usually with respect to the host cell, means that the polynucleotide does not naturally occur in the genome of the host cell or that the polypeptide is not naturally produced by that cell.
- the invention features cells, e.g., transformed host cells or recombinant host cells that contain a nucleic acid encompassed by the invention.
- a "transformed cell” or “recombinant cell” is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid according to the invention.
- Both prokaryotic and eukaryotic cells are included, e.g., bacteria, fungi, yeast, and the like, especially preferred are cells from filamentous fungi, such as Aspergillus niger.
- a host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein.
- Various host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products.
- Appropriate cell lines or host systems familiar to those of skill in the art of molecular biology and/or microbiology can be chosen to ensure the desired and correct modification and processing of the foreign protein expressed.
- eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.
- Such host cells are well known in the art.
- a cell as described above may be used to in the preparation of a polypeptide according to the invention.
- Such a method typically comprises cultivating a host cell (e. g. transformed or transfected with an expression vector as described above) under conditions to provide for expression (by the vector) of a coding sequence encoding the polypeptide, and optionally recovering the expressed polypeptide.
- a host cell e. g. transformed or transfected with an expression vector as described above
- Polynucleotides of the invention can be incorporated into a recombinant replicable vector, e. g. an expression vector.
- the vector may be used to replicate the nucleic acid in a compatible host cell.
- the invention provides a method of making a polynucleotide of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about the replication of the vector.
- the vector may be recovered from the host cell.
- the polypeptide is produced as a secreted protein in which case the nucleotide sequence encoding a mature form of the polypeptide in the expression construct is operably linked to a nucleotide sequence encoding a signal sequence.
- the signal sequence is native (homologous) to the nucleotide sequence encoding the polypeptide.
- the signal sequence is foreign (heterologous) to the nucleotide sequence encoding the polypeptide, in which case the signal sequence is preferably endogenous to the host cell in which the nucleotide sequence according to the invention is expressed. Examples of suitable signal sequences for yeast host cells are the signal sequences derived from yeast a-factor genes.
- a suitable signal sequence for filamentous fungal host cells is e.g. a signal sequence derived from a filamentous fungal amyloglucosidase (AG) gene, e.g. the A. niger glaA gene. This may be used in combination with the amyloglucosidase (also called (gluco) amylase) promoter itself, as well as in combination with other promoters. Hybrid signal sequences may also be used with the context of the present invention.
- AG filamentous fungal amyloglucosidase
- A. niger glaA gene e.g. the A. niger glaA gene.
- This may be used in combination with the amyloglucosidase (also called (gluco) amylase) promoter itself, as well as in combination with other promoters.
- Hybrid signal sequences may also be used with the context of the present invention.
- Preferred heterologous secretion leader sequences are those originating from the fungal amyloglucosidase (AG) gene (glaA-both 18 and 24 amino acid versions e.g. from Aspergillus), the ⁇ -factor gene (yeasts e.g. Saccharomyces and Kluyveromyces) or the ⁇ -amylase gene (Bacillus).
- AG fungal amyloglucosidase
- ⁇ -factor gene e.g. Saccharomyces and Kluyveromyces
- Bactus e.g. Saccharomyces and Kluyveromyces
- the vectors may be transformed or transfected into a suitable host cell as described above to provide for expression of a polypeptide of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptide.
- the invention thus provides host cells transformed or transfected with or comprising a polynucleotide or vector of the invention.
- the polynucleotide is carried in a vector for the replication and expression of the polynucleotide.
- the cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.
- a heterologous host may also be chosen wherein the polypeptide of the invention is produced in a form which is substantially free from other cellulose-degrading or hemicellulose degrading enzymes. This may be achieved by choosing a host which does not normally produce such enzymes.
- the invention encompasses processes for the production of the polypeptide of the invention by means of recombinant expression of a DNA sequence encoding the polypeptide.
- the DNA sequence of the invention can be used for gene amplification and/or exchange of expression signals, such as promoters, secretion signal sequences, in order to allow economic production of the polypeptide in a suitable homologous or heterologous host cell.
- a homologous host cell is a host cell which is of the same species or which is a variant within the same species as the species from which the DNA sequence is derived.
- Suitable host cells are preferably prokaryotic microorganisms such as bacteria, or more preferably eukaryotic organisms, for example fungi, such as yeasts or filamentous fungi, or plant cells.
- yeast cells are preferred over fungal cells because they are easier to manipulate.
- some proteins are either poorly secreted from yeasts, or in some cases are not processed properly (e. g. hyperglycosylation in yeast). In these instances, a fungal host organism should be selected.
- the host cell may over-express the polypeptide, and techniques for engineering over-expression are well known.
- the host may thus have two or more copies of the encoding polynucleotide (and the vector may thus have two or more copies accordingly).
- the "parent microbial host cell” and the “mutant microbial host cell” may be any type of host cell.
- the specific embodiments of the mutant microbial host cell are hereafter described. It will be clear to those skilled in the art that embodiments applicable to the mutant microbial host cell are as well applicable to the parent microbial host cell unless otherwise indicated.
- the mutant microbial host cell according to the present invention may be a prokaryotic cell.
- the prokaryotic host cell is bacterial cell.
- the term "bacterial cell” includes both Gram-negative and Gram-positive microorganisms. Suitable bacteria may be selected from e.g. Escherichia, Anabaena, Caulobactert, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacterium, Rhizobium ( Sinorhizobium ), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus or Streptomyces.
- the bacterial cell is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus, G. oxydans, Caulobactert crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pseudomonas zeaxanthinifaciens, Paracoccus denitrificans, E. coli, C. glutamicum, Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium melioti and Rhizobium radiobacter.
- the mutant microbial host cell according to the invention is a eukaryotic host cell.
- the eukaryotic cell is a mammalian, insect, plant, fungal, or algal cell.
- Preferred mammalian cells include e.g. Chinese hamster ovary (CHO) cells, COS cells, 293 cells, PerC6 cells, and hybridomas.
- Preferred insect cells include e.g. Sf9 and Sf21 cells and derivatives thereof. More preferably, the eukaryotic cell is a fungal cell, i.e.
- a yeast cell such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain. More preferably the eukaryotic host cell is a Kluyveromyces lactis, S. cerevisiae, Hansenula polymorpha, Yarrowia lipolytica or Pichia pastoris, or a filamentous fungal cell. Most preferably, the eukaryotic cell is a filamentous fungal cell.
- Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK ).
- the filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.
- Filamentous fungal strains include, but are not limited to, strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Rasamsonia, Thermoascus, Thielavia, Tolypocladium, and Trichoderma.
- Preferred filamentous fungal cells belong to a species of an Acremonium, Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Talaromyces, Rasamsonia, Thielavia, Fusarium or Trichoderma genus, and most preferably a species of Aspergillus niger, Acremonium alabamense, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Talaromyces emersonii, Rasamsonia emersonii, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium oxysporum, Myceliophthora thermophila, Trichoderma reesei, Thielavia terrestris or Penicillium chrysogenum.
- a more preferred host cell belongs to the genus Aspergillus or Rasamsonia, more preferably the host cell belongs to the species Aspergillus niger or Rasamsonia emersonii.
- the host cell according to the invention is an Aspergillus niger host cell, the host cell preferably is CBS 513.88, CBS124.903 or a derivative thereof.
- Useful strains in the context of the present invention may be Aspergillus niger CBS 513.88, CBS124.903, Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 1011, CBS205.89, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892, P. chrysogenum CBS 455.95, P.
- the mutant microbial host cell when the mutant microbial host cell according to the invention is a filamentous fungal host cell, the mutant microbial host cell may comprise one or more modifications in its genome such that the mutant microbial host cell is deficient in the production of at least one product selected from glucoamylase (glaA), acid stable alpha-amylase (amyA), neutral alpha-amylase (amyBI and amyBII), oxalic acid hydrolase (oahA), a toxin, preferably ochratoxin and/or fumonisin, a protease transcriptional regulator prtT, PepA, a product encoded by the gene hdfA and/or hdfB, a non-ribosomal peptide synthase nps E if compared to a parent host cell and measured under the same conditions.
- glucoamylase glucoamylase
- amyA acid stable alpha-amylase
- the mutant microbial host cell according to the invention when the mutant microbial host cell according to the invention is a filamentous fungal host cell the host cell may comprise one or more modifications in its genome to result in a deficiency in the production of the major extracellular aspartic protease PepA.
- the host cell according to the invention may further comprise a disruption of the pepA gene encoding the major extracellular aspartic protease PepA.
- the mutant microbial host cell according to the invention is a filamentous fungal host cell
- the host cell according to the invention may additionally comprises one or more modifications in its genome to result in a deficiency in the production of the product encoded by the hdf A and/or hdfB gene.
- the host cell according to the invention may further comprise a disruption of the hdfA and/or hdfB gene. Filamentous fungal host cells which are deficient in a product encoded by the hdfA and/or hdfB gene have been described in WO 2005/095624 .
- the mutant microbial host cell according to the invention is a filamentous fungal host cell
- the host cell according to the invention may additionally comprise a modification in its genome which results in the deficiency in the production of the non-ribosomal peptide synthase nps E .
- Such host cells deficient in the production of non-ribosomal peptide synthase npsE have been described in WO2012/001169 (npsE has a genomic sequence as depicted in SEQ ID NO: 35, a coding sequence depicted in SEQ ID NO: 36, the mRNA depicted in SEQ ID NO: 37 and the nrps protein depicted in SEQ ID NO: 38 of WO2012/001169 ).
- the mutant microbial host cell according to the invention is a filamentous fungal host cell
- the host cell may additionally comprise at least two substantially homologous DNA domains suitable for integration of one or more copies of a polynucleotide encoding a compound of interest wherein at least one of the at least two substantially homologous DNA domains is adapted to have enhanced integration preference for the polynucleotide encoding a compound of interest compared to the substantially homologous DNA domain it originates from, and wherein the substantially homologous DNA domain where the adapted substantially homologous DNA domain originates from has a gene conversion frequency that is at least 10% higher than one of the other of the at least two substantially homologous DNA domains.
- Strains containing two or more copies of these substantially homologous DNA domains are also referred hereafter as strain containing two or more amplicons.
- Examples of host cells comprising such amplicons are e.g. described in van Dijck et al, 2003, Regulatory Toxicology and Pharmacology 28; 27-35 : On the safety of a new generation of DSM Aspergillus niger enzyme production strains. In van Dijck et al, an Aspergillus niger strain is described that comprises 7 amplified glucoamylase gene loci, i.e. 7 amplicons.
- Preferred host cells within this context are filamentous fungus host cells, preferably A.
- niger host cells comprising two or more amplicons, preferably two or more ⁇ gla A amplicons (preferably comprising 3, 4, 5, 6, 7 ⁇ gla A amplicons) wherein the amplicon which has the highest frequency of gene conversion, has been adapted to have enhanced integration preference for the polynucleotide encoding a compound of interest compared to the amplicon it originates from.
- Adaptation of the amplicon can be performed according to any one of the methods described in WO2011/009700 .
- host cells comprising three ⁇ gla A amplicons being a Bam HI truncated amplicon, a Sal I truncated amplicon and a Bgl II truncated amplicon and wherein the Bam HI amplicon has been adapted to have enhanced integration preference for a polynucleotide encoding a compound of interest compared to the Bam HI amplicon it originates from.
- Host cells comprising two or more amplicons wherein one amplicon has been adapted to have enhanced integration preference for a polynucleotide encoding a compound of interest compared to the amplicon it originates from are hereafter referred as host cells comprising an adapted amplicon.
- the host cell according to the invention may additionally comprises a modification of Sec61.
- a preferred SEC61 modification is a modification which results in a one-way mutant of SEC61; i.e. a mutant wherein the de novo synthesized protein can enter the ER via SEC61, but the protein cannot leave the ER via SEC61.
- Such modifications are extensively described in WO2005/123763 .
- the SEC 61 modification is the S376W mutation in which Serine 376 is replaced by Tryptophan.
- Host cells according to the invention include plant cells, and the invention therefore extends to transgenic organisms, such as plants and parts thereof, which contain one or more cells of the invention.
- the cells may heterologously express the polypeptide of the invention or may heterologously contain one or more of the polynucleotides of the invention.
- the transgenic (or genetically modified) plant may therefore have inserted (e. g. stably) into its genome a sequence encoding one or more of the polypeptides of the invention.
- the transformation of plant cells can be performed using known techniques, for example using a Ti or a Ri plasmid from Agrobacterium tumefaciens.
- the plasmid (or vector) may thus contain sequences necessary to infect a plant, and derivatives of the Ti and/or Ri plasmids may be employed.
- a part of a plant such as a leaf, root or stem
- the plant to be infected can be wounded, for example by cutting the plant with a razor or puncturing the plant with a needle or rubbing the plant with an abrasive.
- the wound is then inoculated with the Agrobacterium.
- the plant or plant part can then be grown on a suitable culture medium and allowed to develop into a mature plant.
- Regeneration of transformed cells into genetically modified plants can be achieved by using known techniques, for example by selecting transformed shoots using an antibiotic and by sub-culturing the shoots on a medium containing the appropriate nutrients, plant hormones and the like.
- the invention also includes cells that have been modified to express the cellobiohydrolase of the invention or a variant thereof.
- Such cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, lower eukaryotic cells, such as yeast and (e. g. filamentous) fungal cells or prokaryotic cells such as bacterial cells.
- proteins of the invention can be transiently expressed in a cell line or on a membrane, such as for example in a baculovirus expression system.
- a cell line or on a membrane such as for example in a baculovirus expression system.
- Such systems which are adapted to express the proteins according to the invention, are also included within the scope of the present invention.
- the production of the polypeptide of the invention can be effected by the culturing of microbial expression hosts, which have been transformed with one or more polynucleotides of the present invention, in a conventional nutrient fermentation medium.
- the recombinant host cells according to the invention may be cultured using procedures known in the art. For each combination of a promoter and a host cell, culture conditions are available which are conducive to the expression the DNA sequence encoding the polypeptide. After reaching the desired cell density or titer of the polypeptide the culture is stopped and the polypeptide is recovered using known procedures.
- the fermentation medium can comprise a known culture medium containing a carbon source (e.g. glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellolytic biomass hydrolysate, etc.), a nitrogen source (e.g. ammonium sulphate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e.g. yeast extract, malt extract, peptone, etc.) and inorganic nutrient sources (e.g. phosphate, magnesium, potassium, zinc, iron, etc.).
- a inducer e.g. cellulose, pectin, xylan, maltose, maltodextrin or xylogalacturonan
- an inducer e.g. cellulose, pectin, xylan, maltose, maltodextrin or xylogalacturonan
- an inducer e.g. cellulose, pectin
- the selection of the appropriate medium may be based on the choice of expression host and/or based on the regulatory requirements of the expression construct. Such media are known to those skilled in the art.
- the medium may, if desired, contain additional components favoring the transformed expression hosts over other potentially contaminating microorganisms.
- the fermentation can be performed over a period of from about 0.5 to about 30 days. It may be a batch, continuous or fed-batch process, suitably at a temperature in the range of 0-100°C or 0-80°C, for example, from about 0 to about 50°C and/or at a pH, for example, from about 2 to about 10.
- Preferred fermentation conditions are a temperature in the range of from about 20 to about 45°C and/or at a pH of from about 3 to about 9. The appropriate conditions are usually selected based on the choice of the expression host and the protein to be expressed.
- the cells can be removed from the fermentation broth by means of centrifugation or filtration. After fermentation has stopped or after removal of the cells, the polypeptide of the invention may then be recovered and, if desired, purified and isolated by conventional means.
- the invention provides a composition comprising a polypeptide of the invention and a cellulase and/or a hemicellulase and/or a pectinase and/or ligninase or a lignin-modifying enzyme.
- a composition of the invention will typically comprise a hemicellulase and/or a pectinase and/or ligninase or a lignin-modifying enzyme in addition to the polypeptide of the invention.
- composition of the invention will typically comprise a cellulase and/or a pectinase and/or ligninase or a lignin-modifying enzyme in addition to the polypeptide of the invention.
- composition of the invention will typically comprise a cellulase and/or a hemicellulase and/or ligninase or a lignin-modifying enzyme in addition to the polypeptide of the invention.
- composition of the invention will typically comprise a cellulase and/or a hemicellulase and/or a pectinase in addition to the polypeptide of the invention.
- a composition of the invention may comprise one, two or three or more classes of cellulase, for example one, two or all of a GH61, an endo-1,4- ⁇ -glucanase (EG), an exo-cellobiohydrolase (CBH) and a ⁇ -glucosidase (BGL).
- a GH61 an endo-1,4- ⁇ -glucanase
- CBH exo-cellobiohydrolase
- BGL ⁇ -glucosidase
- a composition of the invention may comprise a polypeptide which has the same enzymatic activity, for example the same type of cellulase, hemicellulase and/or pectinase activity as that provided by a polypeptide of the invention.
- a composition of the invention may comprise a polypeptide which has a different type of cellulase activity and/or hemicellulase activity and/or pectinase activity than that provided by a polypeptide of the invention.
- a composition of the invention may comprise one type of cellulase and/or hemicellulase activity and/or pectinase activity provided by a polypeptide of the invention and a second type of cellulase and/or hemicellulase activity and/or pectinase activity provided by an additional hemicellulase/pectinase.
- a cellulase is any polypeptide which is capable of degrading or cellulose.
- a polypeptide which is capable of degrading cellulose is one which is capable of catalysing the process of breaking down cellulose into smaller units, either partially, for example into cellodextrins, or completely into glucose monomers.
- a cellulase according to the invention may give rise to a mixed population of cellodextrins and glucose monomers when contacted with the cellulase. Such degradation will typically take place by way of a hydrolysis reaction.
- a hemicellulase is any polypeptide which is capable of degrading or hemicellulose. That is to say, a hemicellulase may be capable of degrading or one or more of xylan, glucuronoxylan, arabinoxylan, glucomannan and xyloglucan.
- a polypeptide which is capable of degrading a hemicellulose is one which is capable of catalysing the process of breaking down the hemicellulose into smaller polysaccharides, either partially, for example into oligosaccharides, or completely into sugar monomers, for example hexose or pentose sugar monomers.
- a hemicellulase according to the invention may give rise to a mixed population of oligosaccharides and sugar monomers when contacted with the hemicellulase. Such degradation will typically take place by way of a hydrolysis reaction.
- a pectinase is any polypeptide which is capable of degrading or pectin.
- a polypeptide which is capable of degrading pectin is one which is capable of catalysing the process of breaking down pectin into smaller units, either partially, for example into oligosaccharides, or completely into sugar monomers.
- a pectinase according to the invention may give rise to a mixed population of oligosaccharides and sugar monomers when contacted with the pectinase. Such degradation will typically take place by way of a hydrolysis reaction.
- a ligninase or a lignin-modifying enzyme is any polypeptide which is capable of degrading or modifying lignin or degradation components thereof.
- a polypeptide which is capable of degrading or modifying lignin is one which is capable of catalysing the process of breaking down lignin into smaller units, either partially, for example into monophenolic compounds.
- a ligninase or a lignin-modifying enzyme according to the invention may give rise to a mixed population of phenolic compounds when contacted with the lignin. Such degradation will typically take place by way of an oxidation reaction.
- a ligninase or a lignin-modifying enzyme may also be any polypeptide which is capable of degrading phenolic degradation products of lignin.
- a polypeptide which is capable of degrading phenolic degradation products of lignin is one which is capable of catalysing the process of breaking down phenolic degradation products of lignin into even smaller units, for example by catalysing a ring opening reaction of the phenolic ring.
- a ligninase or a lignin-modifying enzyme according to the invention may give rise to a mixed population of ring-opened degradation products of phenolic compounds when contacted with the phenolic degradation products of lignin.
- the a ligninase or a lignin-modifying enzyme may further be capable of breaking linkages between cellulose or hemicellulose and the lignin or degradation products thereof.
- Enzymes that can break down lignin include lignin peroxidases, manganese peroxidases, laccases and feruloyl esterases, and other enzymes described in the art known to depolymerize or otherwise break lignin polymers. Also included are enzymes capable of hydrolyzing bonds formed between hemicellulosic sugars (notably arabinose) and lignin.
- Ligninases include but are not limited to the following group of enzymes: lignin peroxidases (EC 1.11.14), manganese peroxidases (EC 1.11.1.13), laccases (EC 1.10.3.2) and feruloyl esterases (EC 3.1.1.73).
- composition of the invention may comprise any cellulase, for example, a GH61, a cellobiohydrolase, an endo- ⁇ -1,4-glucanase, a ⁇ -glucosidase or a ⁇ -(1,3)(1,4)-glucanase.
- a cellulase for example, a GH61, a cellobiohydrolase, an endo- ⁇ -1,4-glucanase, a ⁇ -glucosidase or a ⁇ -(1,3)(1,4)-glucanase.
- GH61 glycoside hydrolase family 61 or sometimes referred to EGIV proteins are oxygen-dependent polysaccharide monooxygenases (PMO's) according to the latest literature. Often in literature these proteins are mentioned to enhance the action of cellulases on lignocellulose substrates. GH61 was originally classified as endogluconase based on measurement of very weak endo-1,4- ⁇ -d-glucanase activity in one family member. The term "GH61" as used herein, is to be understood as a family of enzymes, which share common conserved sequence portions and foldings to be classified in family 61 of the well-established CAZY GH classification system (http://www.cazy.org/GH61.html). The glycoside hydrolase family 61 is a member of the family of glycoside hydrolases EC 3.2.1. GH61 is used herein as being part of the cellulases.
- PMO's oxygen-dependent polysaccharide monooxygenases
- a cellobiohydrolase (EC 3.2.1.91) is any polypeptide which is capable of catalysing the hydrolysis of 1,4- ⁇ -D-glucosidic linkages in cellulose or cellotetraose, releasing cellobiose from the non-reducing ends of the chains.
- This enzyme may also be referred to as cellulase 1,4- ⁇ -cellobiosidase, 1,4- ⁇ -cellobiohydrolase, 1,4- ⁇ -D-glucan cellobiohydrolase, avicelase, exo-1,4- ⁇ -D-glucanase, exocellobiohydrolase or exoglucanase. It may be a have the EC code EC 3.2.1.91.
- an endo- ⁇ -1,4-glucanase (EC 3.2.1.4) is any polypeptide which is capable of catalysing the endohydrolysis of 1,4-3-D-glucosidic linkages in cellulose, lichenin or cereal ⁇ -D-glucans. Such a polypeptide may also be capable of hydrolyzing 1,4-linkages in ⁇ -D-glucans also containing 1,3-linkages.
- This enzyme may also be referred to as cellulase, avicelase, ⁇ -1,4-endoglucan hydrolase, ⁇ -1,4-glucanase, carboxymethyl cellulase, celludextrinase, endo-1,4- ⁇ -D-glucanase, endo-1,4- ⁇ -D-glucanohydrolase, endo-1,4- ⁇ -glucanase or endoglucanase.
- the endo-glucanase may also catalyze the cleavage of xyloglucan, a backbone of ⁇ 1 ⁇ 4-linked glucose residues, most of which substituted with 1-6 linked xylose side chains, and the enzyme is then referred to as a xyloglucan-specific endo- ⁇ -1,4-glucanase or a xyloglucanase.
- a ⁇ -glucosidase (EC 3.2.1.21) is any polypeptide which is capable of catalysing the hydrolysis of terminal, non-reducing ⁇ -D-glucose residues with release of ⁇ -D-glucose.
- Such a polypeptide may have a wide specificity for ⁇ -D-glucosides and may also hydrolyze one or more of the following: a ⁇ -D-galactoside, an ⁇ -L-arabinoside, a ⁇ -D-xyloside or a ⁇ -D-fucoside.
- This enzyme may also be referred to as amygdalase, ⁇ -D-glucoside glucohydrolase, cellobiase or gentobiase.
- a ⁇ -(1,3)(1,4)-glucanase (EC 3.2.1.73) is any polypeptide which is capable of catalyzing the hydrolysis of 1,4- ⁇ -D-glucosidic linkages in ⁇ -D-glucans containing 1,3- and 1,4-bonds.
- Such a polypeptide may act on lichenin and cereal ⁇ -D-glucans, but not on ⁇ -D-glucans containing only 1,3- or 1,4-bonds.
- This enzyme may also be referred to as licheninase, 1,3-1,4- ⁇ -D-glucan 4-glucanohydrolase, ⁇ -glucanase, endo- ⁇ -1,3-1,4 glucanase, lichenase or mixed linkage ⁇ -glucanase.
- An alternative for this type of enzyme is EC 3.2.1.6, which is described as endo-1,3(4)-beta-glucanase.
- This type of enzyme hydrolyses 1,3- or 1,4-linkages in beta-D-glucans when the glucose residue whose reducing group is involved in the linkage to be hydrolyzed is itself substituted at C-3.
- Alternative names include endo-1,3-beta-glucanase, laminarinase, 1,3-(1,3;1,4)-beta-D-glucan 3 (4) glucanohydrolase; substrates include laminarin, lichenin and cereal beta-D-glucans.
- a composition of the invention may comprise any hemicellulase, for example, an endo-xylanase, a ⁇ -xylosidase, a ⁇ -L-arabionofuranosidase, an ⁇ -D-glucuronidase, an cellobiohydrolase, a feruloyl esterase, a coumaroyl esterase, an ⁇ -galactosidase, a ⁇ -galactosidase, a ⁇ -mannanase or a ⁇ -mannosidase.
- hemicellulase for example, an endo-xylanase, a ⁇ -xylosidase, a ⁇ -L-arabionofuranosidase, an ⁇ -D-glucuronidase, an cellobiohydrolase, a feruloyl esterase, a coumaroyl esterase, an ⁇ -galactosidase,
- an endoxylanase (EC 3.2.1.8) is any polypeptide which is capable of catalyzing the endo-hydrolysis of 1,4- ⁇ -D-xylosidic linkages in xylans.
- This enzyme may also be referred to as endo-1,4- ⁇ -xylanase or 1,4- ⁇ -D-xylan xylanohydrolase.
- An alternative is EC 3.2.1.136, a glucuronoarabinoxylan endoxylanase, an enzyme that is able to hydrolyze 1,4 xylosidic linkages in glucuronoarabinoxylans.
- a ⁇ -xylosidase (EC 3.2.1.37; GH3) is any polypeptide which is capable of catalyzing the hydrolysis of 1,4- ⁇ -D-xylans, to remove successive D-xylose residues from the non-reducing termini. Such enzymes may also hydrolyze xylobiose.
- This enzyme may also be referred to as xylan 1,4- ⁇ -xylosidase, 1,4- ⁇ -D-xylan xylohydrolase, exo-1,4- ⁇ -xylosidase or xylobiase.
- an ⁇ -L-arabinofuranosidase (EC 3.2.1.55) is any polypeptide which is capable of acting on ⁇ -L-arabinofuranosides, ⁇ -L-arabinans containing (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans and arabinogalactans.
- This enzyme may also be referred to as ⁇ -N-arabinofuranosidase, arabinofuranosidase or arabinosidase.
- This enzyme may also be referred to as alpha-glucuronidase or alpha-glucosiduronase. These enzymes may also hydrolyze 4-O-methylated glucoronic acid, which can also be present as a substituent in xylans.
- Alternative is EC 3.2.1.131: xylan alpha-1,2-glucuronosidase, which catalyses the hydrolysis of alpha-1,2-(4-O-methyl)glucuronosyl links.
- an acetyl xylan esterase (EC 3.1.1.72) is any polypeptide which is capable of catalyzing the deacetylation of xylans and xylo-oligosaccharides.
- a polypeptide may catalyze the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate or p-nitrophenyl acetate but, typically, not from triacetylglycerol.
- Such a polypeptide typically does not act on acetylated mannan or pectin.
- the saccharide may be, for example, an oligosaccharide or a polysaccharide. It may typically catalyze the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar, which is usually arabinose in 'natural' substrates. p-nitrophenol acetate and methyl ferulate are typically poorer substrates.
- This enzyme may also be referred to as cinnamoyl ester hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. It may also be referred to as a hemicellulase accessory enzyme, since it may help xylanases and pectinases to break down plant cell wall hemicellulose and pectin.
- the saccharide may be, for example, an oligosaccharide or a polysaccharide.
- This enzyme may also be referred to as trans-4-coumaroyl esterase, trans-p-coumaroyl esterase, p-coumaroyl esterase or p-coumaric acid esterase. This enzyme also falls within EC 3.1.1.73 so may also be referred to as a feruloyl esterase.
- an ⁇ -galactosidase (EC 3.2.1.22) is any polypeptide which is capable of catalyzing the hydrolysis of terminal, non-reducing ⁇ -D-galactose residues in ⁇ -D-galactosides, including galactose oligosaccharides, galactomannans, galactans and arabinogalactans. Such a polypeptide may also be capable of hydrolyzing ⁇ -D-fucosides. This enzyme may also be referred to as melibiase.
- a ⁇ -gaiactosidase (EC 3.2.1.23) is any polypeptide which is capable of catalyzing the hydrolysis of terminal non-reducing ⁇ -D-galactose residues in ⁇ -D-galactosides. Such a polypeptide may also be capable of hydrolyzing ⁇ -L-arabinosides.
- This enzyme may also be referred to as exo-(1->4)- ⁇ -D-galactanase or lactase.
- a ⁇ -mannanase (EC 3.2.1.78) is any polypeptide which is capable of catalyzing the random hydrolysis of 1,4- ⁇ -D-mannosidic linkages in mannans, galactomannans and glucomannans.
- This enzyme may also be referred to as mannan endo-1,4- ⁇ -mannosidase or endo-1,4-mannanase.
- a ⁇ -mannosidase (EC 3.2.1.25) is any polypeptide which is capable of catalyzing the hydrolysis of terminal, non-reducing ⁇ -D-mannose residues in ⁇ -D-mannosides.
- This enzyme may also be referred to as mannanase or mannase.
- a composition of the invention may comprise any pectinase, for example an endo polygalacturonase, a pectin methyl esterase, an endo-galactanase, a beta galactosidase, a pectin acetyl esterase, an endo-pectin lyase, pectate lyase, alpha rhamnosidase, an exo-galacturonase, an exo-polygalacturonate lyase, a rhamnogalacturonan hydrolase, a rhamnogalacturonan lyase, a rhamnogalacturonan acetyl esterase, a rhamnogalacturonan galacturonohydrolase or a xylogalacturonase.
- pectinase for example an endo polygalacturonase, a pectin methyl
- an endo-polygalacturonase (EC 3.2.1.15) is any polypeptide which is capable of catalyzing the random hydrolysis of 1,4- ⁇ -D-galactosiduronic linkages in pectate and other galacturonans.
- This enzyme may also be referred to as polygalacturonase pectin depolymerase, pectinase, endopolygalacturonase, pectolase, pectin hydrolase, pectin polygalacturonase, poly- ⁇ -1,4-galacturonide glycanohydrolase, endogalacturonase; endo-D-galacturonase or poly(1,4- ⁇ -D-galacturonide) glycanohydrolase.
- the enzyme may also be known as pectinesterase, pectin demethoxylase, pectin methoxylase, pectin methylesterase, pectase, pectinoesterase or pectin pectylhydrolase.
- an endo-galactanase (EC 3.2.1.89) is any enzyme capable of catalyzing the endohydrolysis of 1,4- ⁇ -D-galactosidic linkages in arabinogalactans.
- the enzyme may also be known as arabinogalactan endo-1,4- ⁇ -galactosidase, endo-1,4- ⁇ -galactanase, galactanase, arabinogalactanase or arabinogalactan 4- ⁇ -D-galactanohydrolase.
- a pectin acetyl esterase is defined herein as any enzyme which has an acetyl esterase activity which catalyzes the deacetylation of the acetyl groups at the hydroxyl groups of GalUA residues of pectin
- an endo-pectin lyase (EC 4.2.2.10) is any enzyme capable of catalyzing the eliminative cleavage of (1 ⁇ 4)- ⁇ -D-galacturonan methyl ester to give oligosaccharides with 4-deoxy-6-O-methyl- ⁇ -D-galact-4-enuronosyl groups at their non-reducing ends.
- the enzyme may also be known as pectin lyase, pectin trans-eliminase; endo-pectin lyase, polymethylgalacturonic transeliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGL or (1 ⁇ 4)-6-O-methyl- ⁇ -D-galacturonan lyase.
- a pectate lyase (EC 4.2.2.2) is any enzyme capable of catalyzing the eliminative cleavage of (1 ⁇ 4)- ⁇ -D-galacturonan to give oligosaccharides with 4-deoxy- ⁇ -D-galact-4-enuronosyl groups at their non-reducing ends.
- the enzyme may also be known polygalacturonic transeliminase, pectic acid transeliminase, polygalacturonate lyase, endopectin methyltranseliminase, pectate transeliminase, endogalacturonate transeliminase, pectic acid lyase, pectic lyase, ⁇ -1,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N, endo- ⁇ -1,4-polygalacturonic acid lyase, polygalacturonic acid lyase, pectin trans-eliminase, polygalacturonic acid trans-eliminase or (1 ⁇ 4)- ⁇ -D-galacturonan lyase.
- an alpha rhamnosidase (EC 3.2.1.40) is any polypeptide which is capable of catalyzing the hydrolysis of terminal non-reducing ⁇ -L-rhamnose residues in ⁇ -L-rhamnosides or alternatively in rhamnogalacturonan.
- This enzyme may also be known as ⁇ -L-rhamnosidase T, ⁇ -L-rhamnosidase N or ⁇ -L-rhamnoside rhamnohydrolase.
- exo-galacturonase (EC 3.2.1.82) is any polypeptide capable of hydrolysis of pectic acid from the non-reducing end, releasing digalacturonate.
- the enzyme may also be known as exo-poly- ⁇ -galacturonosidase, exopolygalacturonosidase or exopolygalacturanosidase.
- the enzyme may also be known as galacturan 1,4- ⁇ -galacturonidase, exopolygalacturonase, poly(galacturonate) hydrolase, exo-D-galacturonase, exo-D-galacturonanase, exo-poly-D-galacturonase or poly(1,4- ⁇ -D-galacturonide) galacturonohydrolase.
- exo-polygalacturonate lyase (EC 4.2.2.9) is any polypeptide capable of catalyzing eliminative cleavage of 4-(4-deoxy- ⁇ -D-galact-4-enuronosyl)-D-galacturonate from the reducing end of pectate, i.e. de-esterified pectin.
- This enzyme may be known as pectate disaccharide-lyase, pectate exo-lyase, exopectic acid transeliminase, exo-pectate lyase, exopolygalacturonic acid-trans-eliminase, PATE, exo-PATE, exo-PGL or (1 ⁇ 4)- ⁇ -D-galacturonan reducing-end-disaccharide-lyase.
- rhamnogalacturonan hydrolase is any polypeptide which is capable of hydrolyzing the linkage between galactosyluronic acid and rhamnopyranosyl in an endo-fashion in strictly alternating rhamnogalacturonan structures, consisting of the disaccharide [(1,2-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].
- rhamnogalacturonan lyase is any polypeptide which is any polypeptide which is capable of cleaving ⁇ -L-Rhap-(1 ⁇ 4)- ⁇ -D-GalpA linkages in an endo-fashion in rhamnogalacturonan by beta-elimination.
- rhamnogalacturonan acetyl esterase is any polypeptide which catalyzes the deacetylation of the backbone of alternating rhamnose and galacturonic acid residues in rhamnogalacturonan.
- rhamnogalacturonan galacturonohydrolase is any polypeptide which is capable of hydrolyzing galacturonic acid from the non-reducing end of strictly alternating rhamnogalacturonan structures in an exo-fashion.
- xylogalacturonase is any polypeptide which acts on xylogalacturonan by cleaving the ⁇ -xylose substituted galacturonic acid backbone in an endo-manner.
- This enzyme may also be known as xylogalacturonan hydrolase.
- an ⁇ -L-arabinofuranosidase (EC 3.2.1.55) is any polypeptide which is capable of acting on ⁇ -L-arabinofuranosides, ⁇ -L-arabinans containing (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans and arabinogalactans.
- This enzyme may also be referred to as ⁇ -N-arabinofuranosidase, arabinofuranosidase or arabinosidase.
- endo-arabinanase (EC 3.2.1.99) is any polypeptide which is capable of catalyzing endohydrolysis of 1,5- ⁇ -arabinofuranosidic linkages in 1,5-arabinans.
- the enzyme may also be known as endo-arabinase, arabinan endo-1,5- ⁇ -L-arabinosidase, endo-1,5- ⁇ -L-arabinanase, endo- ⁇ -1,5-arabanase; endo-arabanase or 1,5- ⁇ -L-arabinan 1,5- ⁇ -L-arabinanohydrolase.
- a composition of the invention will typically comprise at least one cellulase and/or at least one hemicellulase and/or at least one pectinase (one of which is a polypeptide according to the invention).
- a composition of the invention may comprise a cellobiohydrolase, an endoglucanase and/or a ⁇ -glucosidase.
- Such a composition may also comprise one or more hemicellulases and/or one or more pectinases.
- One or more (for example two, three, four or all) of an amylase, a protease, a lipase, a ligninase, a hexosyltransferase or a glucuronidase may be present in a composition of the invention.
- proteases includes enzymes that hydrolyze peptide bonds (peptidases), as well as enzymes that hydrolyze bonds between peptides and other moieties, such as sugars (glycopeptidases). Many proteases are characterized under EC 3.4, and are suitable for use in the invention incorporated herein by reference. Some specific types of proteases include, cysteine proteases including pepsin, papain and serine proteases including chymotrypsins, carboxypeptidases and metalloendopeptidases.
- Lipase includes enzymes that hydrolyze lipids, fatty acids, and acylglycerides, including phospoglycerides, lipoproteins, diacylglycerols, and the like. In plants, lipids are used as structural components to limit water loss and pathogen infection. These lipids include waxes derived from fatty acids, as well as cutin and suberin.
- “Hexosyltransferase” (2.4.1-) includes enzymes which are capable of transferring glycosyl groups, more specifically hexosyl groups. In addition to transfer of a glycosyl-group from a glycosyl-containing donor to another glycosyl-containing compound, the acceptor, the enzymes can also transfer the glycosyl-group to water as an acceptor. This reaction is also known as a hydrolysis reaction, instead of a transfer reaction.
- An example of a hexosyltransferase which may be used in the invention is a ⁇ -glucanosyltransferase. Such an enzyme may be able to catalyze degradation of (1,3)(1,4)glucan and/or cellulose and/or a cellulose degradation product.
- Glucuronidase includes enzymes that catalyze the hydrolysis of a glucoronoside, for example ⁇ -glucuronoside to yield an alcohol.
- Many glucuronidases have been characterized and may be suitable for use in the invention, for example ⁇ -glucuronidase (EC 3.2.1.31), hyalurono-glucuronidase (EC 3.2.1.36), glucuronosyl-disulfoglucosamine glucuronidase (3.2.1.56), glycyrrhizinate ⁇ -glucuronidase (3.2.1.128) or ⁇ -D-glucuronidase (EC 3.2.1.139).
- a composition of the invention may comprise an expansin or expansin-like protein, such as a swollenin (see Salheimo et al., Eur. J. Biochem. 269, 4202-4211, 2002 ) or a swollenin-like protein.
- an expansin or expansin-like protein such as a swollenin (see Salheimo et al., Eur. J. Biochem. 269, 4202-4211, 2002 ) or a swollenin-like protein.
- Expansins are implicated in loosening of the cell wall structure during plant cell growth. Expansins have been proposed to disrupt hydrogen bonding between cellulose and other cell wall polysaccharides without having hydrolytic activity. In this way, they are thought to allow the sliding of cellulose fibers and enlargement of the cell wall. Swollenin, an expansin-like protein contains an N-terminal Carbohydrate Binding Module Family 1 domain (CBD) and a C-terminal expansin-like domain.
- CBD Carbohydrate Binding Module Family 1 domain
- an expansin-like protein or swollenin-like protein may comprise one or both of such domains and/or may disrupt the structure of cell walls (such as disrupting cellulose structure), optionally without producing detectable amounts of reducing sugars.
- a composition of the invention may comprise the polypeptide product of a cellulose integrating protein, scaffoldin or a scaffoldin-like protein, for example CipA or CipC from Clostridium thermocellum or Clostridium cellulolyticum respectively.
- Scaffoldins and cellulose integrating proteins are multi-functional integrating subunits which may organize cellulolytic subunits into a multi-enzyme complex. This is accomplished by the interaction of two complementary classes of domain, i.e. a cohesion domain on scaffoldin and a dockerin domain on each enzymatic unit.
- the scaffoldin subunit also bears a cellulose-binding module (CBM) that mediates attachment of the cellulosome to its substrate.
- CBM cellulose-binding module
- a scaffoldin or cellulose integrating protein for the purposes of this invention may comprise one or both of such domains.
- a composition of the invention may comprise a cellulose induced protein or modulating protein, for example as encoded by cip1 or cip2 gene or similar genes from Trichoderma reesei / Hypocrea jacorina (see Foreman et al., J. Biol. Chem. 278(34), 31988-31997, 2003 ).
- the polypeptide product of these genes are bimodular proteins, which contain a cellulose binding module and a domain which function or activity can not be related to known glycosyl hydrolase families. Yet, the presence of a cellulose binding module and the co-regulation of the expression of these genes with cellulases components indicates previously unrecognized activities with potential role in biomass degradation.
- a composition of the invention may be composed of a member of each of the classes of the polypeptides mentioned above, several members of one polypeptide class, or any combination of these polypeptide classes.
- a composition of the invention may be composed of polypeptides, for example enzymes, from (1) commercial suppliers; (2) cloned genes expressing polypeptides, for example enzymes; (3) complex broth (such as that resulting from growth of a microbial strain in media, wherein the strains secrete proteins and enzymes into the media; (4) cell lysates of strains grown as in (3); and/or (5) plant material expressing polypeptides, for example enzymes.
- Different polypeptides, for example enzymes in a composition of the invention may be obtained from different sources.
- polypeptides and polypeptide compositions according to the invention may be used in many different applications. For instance, they may be used to produce fermentable sugars. The fermentable sugars can then, as part of a biofuel process, be converted into biogas or ethanol, butanol, isobutanol, 2 butanol or other suitable substances. Alternatively, the polypeptides and their compositions may be used as enzyme, for instance in production of food products, in detergent compositions, in the paper and pulp industry and in antibacterial formulations, in pharmaceutical products such as throat lozenges, toothpastes, and mouthwash. Some of the uses will be illustrated in more detail below.
- compositions described above may be provided concomitantly (i.e. as a single composition per se) or separately or sequentially.
- the invention also relates to the use of the cellobiohydrolase according to the invention and compositions comprising such an enzyme in industrial processes.
- the cellobiohydrolase according to the invention may feature a number of significant advantages over enzymes currently used. Depending on the specific application, these advantages may include aspects such as lower production costs, higher specificity towards the substrate, reduced antigenicity, fewer undesirable side activities, higher yields when produced in a suitable microorganism, more suitable pH and temperature ranges, non-inhibition by hydrophobic, lignin-derived products or less product inhibition or, in the case of the food industry a better taste or texture of a final product as well as food grade and kosher aspects.
- a cellobiohydrolase or composition of the invention may be used in any process which requires the treatment of a material which comprises polysaccharide.
- a polypeptide or composition of the invention may be used in the treatment of polysaccharide material.
- polysaccharide material is a material which comprises or consists essential of one or, more typically, more than one polysaccharide.
- plants and material derived therefrom comprise significant quantities of non-starch polysaccharide material. Accordingly, a polypeptide of the invention may be used in the treatment of a plant or fungal material or a material derived therefrom.
- lignocellulose also referred to herein as lignocellulolytic biomass
- Lignocellulose is plant material that comprises cellulose and hemicellulose and lignin.
- the carbohydrate polymers (cellulose and hemicelluloses) are tightly bound to the lignin by hydrogen and covalent bonds.
- a polypeptide of the invention may be used in the treatment of lignocellulolytic material.
- lignocellulolytic material is a material which comprises or consists essential of lignocellulose.
- the non-starch polysaccharide may be a lignocellulosic material/biomass.
- the invention provides a method of treating a substrate comprising non-starch polysaccharide in which the treatment comprises the degradation and/or hydrolysis and/or modification of cellulose and/or hemicellulose and/or a pectic substance.
- Degradation in this context indicates that the treatment results in the generation of hydrolysis products of cellulose and/or hemicellulose and/or a pectic substance, i.e. saccharides of shorter length are present as result of the treatment than are present in a similar untreated non-starch polysaccharide.
- degradation in this context may result in the liberation of oligosaccharides and/or sugar monomers.
- said substrate may be provided in the form of a plant or a plant derived material or a material comprising a plant or plant derived material, for example a plant pulp, a plant extract, a foodstuff or ingredient therefore, a fabric, a textile or an item of clothing.
- Lignocellulolytic biomass suitable for use in the invention includes biomass and can include virgin biomass and/or non-virgin biomass such as agricultural biomass, commercial organics, construction and demolition debris, municipal solid waste, waste paper and yard waste.
- biomass include trees, shrubs and grasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks, corn cobs, corn kernel including fiber from kernels, products and by-products from milling of grains such as corn, wheat and barley (including wet milling and dry milling) often called "bran or fiber" as well as municipal solid waste, waste paper and yard waste.
- the biomass can also be, but is not limited to, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, and pulp and paper mill residues.
- Agricultural biomass includes branches, bushes, canes, corn and corn husks, energy crops, forests, fruits, flowers, grains, grasses, herbaceous crops, leaves, bark, needles, logs, roots, saplings, short rotation woody crops, shrubs, switch grasses, trees, vegetables, fruit peels, vines, sugar beet pulp, wheat middlings, oat hulls, and hard and soft woods (not including woods with deleterious materials).
- agricultural biomass includes organic waste materials generated from agricultural processes including farming and forestry activities, specifically including forestry wood waste. Agricultural biomass may be any of the aforestated singularly or in any combination or mixture thereof.
- biomass are orchard primings, chaparral, mill waste, urban wood waste, municipal waste, logging waste, forest thinnings, short- rotation woody crops, industrial waste, wheat straw, oat straw, rice straw, barley straw, rye straw, flax straw, soy hulls, rice hulls, rice straw, corn gluten feed, oat hulls, sugar cane, corn stover, corn stalks, corn cobs, corn husks, prairie grass, gamagrass, foxtail; sugar beet pulp, citrus fruit pulp, seed hulls, cellulosic animal wastes, lawn clippings, cotton, seaweed, trees, shrubs, grasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks, corn hobs, corn kernel, fiberfrom kernels, products and by-products from wet or dry milling of grains, municipal solid waste, waste paper, yard waste, herbaceous material, agricultural residues, forestry residues, municipal solid waste, waste paper, pulp, paper mill
- biomass/feedstock may additionally be pretreated with heat, mechanical and/or chemical modification or any combination of such methods in order to enhance enzymatic degradation.
- the feedstock may optionally be pre-treated with heat, mechanical and/or chemical modification or any combination of such methods in order to to enhance the accessibility of the substrate to enzymatic hydrolysis and/or hydrolyse the hemicellulose and/or solubilize the hemicellulose and/or cellulose and/or lignin, in any way known in the art.
- the pretreatment may comprise exposing the lignocellulosic material to (hot) water, steam (steam explosion), an acid, a base, a solvent, heat, a peroxide, ozone, mechanical shredding, grinding, milling or rapid depressurization, or a combination of any two or more thereof.
- This chemical pretreatment is often combined with heat-pretreatment, e.g. between 150-220 °C for 1 to 30 minutes.
- a liquefaction/hydrolysis or presaccharification step involving incubation with an enzyme or enzyme mixture can be utilized.
- the presaccharification step can be performed at many different temperatures but it is preferred that the presaccharification step occur at the temperature best suited to the enzyme mix being tested, or the predicted enzyme optimum of the enzymes to be tested.
- the temperature of the presaccharification step may range from about 10°C to about 95 °C, about 20 °C to about 85 °C, about 30 °C to about 70 °C, about 40 °C to about 60 °C, about 37 °C to about 50 °C, preferably about 37 °C to about 80 °C, more preferably about 60-70°C even more preferably around 65° C.
- the pH of the presaccharification mixture may range from about 2.0 to about 10.0, but is preferably about 3.0 to about 7.0, more preferably about 4.0 to about 6.0, even more preferably about 4.0 to about 5.0. Again, the pH may be adjusted to maximize enzyme activity and may be adjusted with the addition of the enzyme. Comparison of the results of the assay results from this test will allow one to modify the method to best suit the enzymes being tested.
- the liquefaction/hydrolysis or presaccharification step reaction may occur from several minutes to several hours, such as from about 1 hour to about 120 hours, preferably from about 2 hours to about 48 hours, more preferably from about 2 to about 24 hours, most preferably for from about 2 to about 6 hours.
- the cellulase treatment may occur from several minutes to several hours, such as from about 6 hours to about 120 hours, preferably about 12 hours to about 72 hours, more preferably about 24 to 48 hours.
- the invention provides a method for producing a sugar from a lignocellulosic material which method comprises contacting a polypeptide of the invention to a composition of the invention with the lignocellulosic material.
- Such a method allows free sugars (monomers) and/or oligosaccharides to be generated from lignocellulosic biomass. These methods involve converting lignocellulosic biomass to free sugars and small oligosaccharides with a polypeptide or composition of the invention.
- a complex carbohydrate such as lignocellulose into sugars preferably allows conversion into fermentable sugars.
- Such a process may be referred to as "saccharification.”
- a method of the invention may result in the liberation of one or more hexose and/or pentose sugars, such as one or more of glucose, xylose, arabinose, galactose, galacturonic acid, glucuronic acid, mannose, rhamnose, ribose and fructose.
- another aspect of the invention includes methods that utilize the polypeptide of composition of the invention described above together with further enzymes or physical treatments such as temperature and pH to convert the lignocellulosic plant biomass to sugars and oligosaccharides.
- composition has been discussed as a single mixture it is recognized that the enzymes may be added sequentially where the temperature, pH, and other conditions may be altered to increase the activity of each individual enzyme. Alternatively, an optimum pH and temperature can be determined for the enzyme mixture.
- enzymes are reacted with substrate under any appropriate conditions.
- enzymes can be incubated at about 25°C, about 30 °C, about 35 °C, about 37 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C or higher. That is, they can be incubated at a temperature of from about 20°C to about 95 °C, for example in buffers of low to medium ionic strength and/or from low to neutral pH.
- the buffer has an ion concentration of about 200 millimolar (mM) or less for any single ion component.
- the pH may range from about pH 2.5, about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5, about pH 5.5, about pH 6, about pH 6.5, about pH 7, about pH 7.5, about pH 8.0, to about pH 8.5.
- the pH range will be from about pH 3.0 to about pH 7.
- Incubation of enzyme combinations under these conditions results in release or liberation of substantial amounts of the sugar from the lignocellulose.
- substantial amount is intended at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of available sugar.
- the polypeptides can be produced either exogenously in microorganisms, yeasts, fungi, bacteria or plants, then isolated and added, for example, to lignocellulosic feedstock.
- the enzymes are produced, but not isolated, and crude cell mass fermentation broth, or plant material (such as corn stover), and the like may be added to, for example, the feedstock.
- the crude cell mass or enzyme production medium or plant material may be treated to prevent further microbial growth (for example, by heating or addition of antimicrobial agents), then added to, for example, a feedstock.
- These crude enzyme mixtures may include the organism producing the enzyme.
- the enzyme may be produced in a fermentation that uses feedstock (such as corn stover) to provide nutrition to an organism that produces an enzyme(s).
- feedstock such as corn stover
- plants that produce the enzymes may themselves serve as a lignocellulosic feedstock and be added into lignocellulosic feedstock.
- the fermentable sugars can be converted to useful value-added fermentation products, non-limiting examples of which include amino acids, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels, or other organic polymers, lactic acid, and ethanol, including fuel ethanol.
- the sugars may be used as feedstocks for fermentation into chemicals, plastics, such as for instance succinic acid and (bio) fuels, including ethanol, methanol, butanol synthetic liquid fuels and biogas.
- an enzyme or combination of enzymes acts on a lignocellulosic substrate or plant biomass, serving as the feedstock, so as to convert this complex substrate to simple sugars and oligosaccharides for the production of ethanol or other useful fermentation products.
- Sugars released from biomass can be converted to useful fermentation products such a one of those including, but not limited to, amino acids, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, and ethanol, including fuel ethanol.
- the invention provides a method for the preparation of a fermentation product, which method comprises:
- the fermentation may be carried out under aerobic or anaerobic conditions.
- the process is carried out under micro-aerophilic or oxygen limited conditions.
- An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably about 5 or less, about 2.5 or less or about 1 mmol/L/h or less, and wherein organic molecules serve as both electron donor and electron acceptors.
- An oxygen-limited fermentation process is a process in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid.
- the degree of oxygen limitation is determined by the amount and composition of the ingoing gas flow as well as the actual mixing/mass transfer properties of the fermentation equipment used.
- the rate of oxygen consumption is at least about 5.5, more preferably at least about 6 and even more preferably at least about 7 mmol/L/h.
- a method for the preparation of a fermentation product may optionally comprise recovery of the fermentation product.
- Fermentation and Saccharification may also be executed in Simultaneous Saccharification and Fermentation (SSF) mode.
- SSF Simultaneous Saccharification and Fermentation
- One of the advantages of this mode is reduction of the sugar inhibition on enzymatic hydrolysis (Sugar inhibition on cellulases is described by Caminal B&B Vol XXVII Pp 1282-1290 ).
- Fermentation products which may be produced according to the invention include amino acids, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels, or other organic polymers, lactic acid, and ethanol, including fuel ethanol (the term "ethanol” being understood to include ethyl alcohol or mixtures of ethyl alcohol and water).
- Specific value-added products that may be produced by the methods of the invention include, but not limited to, biofuels (including ethanol and butanol and a biogas); lactic acid; a plastic; a specialty chemical; an organic acid, including citric acid, succinic acid, fumaric acid, itaconic acid and maleic acid; 3-hydoxy-propionic acid, acrylic acid; acetic acid; 1,3-propane-diol; ethylene, glycerol; a solvent; an animal feed supplement; a pharmaceutical, such as a ⁇ -lactam antibiotic or a cephalosporin; vitamins; an amino acid, such as lysine, methionine, tryptophan, threonine, and aspartic acid; an industrial enzyme, such as a protease, a cellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, an oxidore
- Biogas typically refers to a gas produced by the biological breakdown of organic matter, for example non-starch carbohydrate containing material, in the absence of oxygen.
- Biogas originates from biogenic material and is a type of biofuel.
- One type of biogas is produced by anaerobic digestion or fermentation of biodegradable materials such as biomass, manure or sewage, municipal waste, and energy crops.
- This type of biogas is comprised primarily of methane and carbon dioxide.
- the gas methane can be combusted or oxidized with oxygen.
- Air contains 21% oxygen. This energy release allows biogas to be used as a fuel.
- Biogas can be used as a low-cost fuel in any country for any heating purpose, such as cooking. It can also be utilized in modern waste management facilities where it can be used to run any type of heat engine, to generate either mechanical or electrical power.
- the first step in microbial biogas production consists in the enzymatic degradation of polymers and complex substrates (for example non-starch carbohydrate).
- the invention provides a method for preparation of a biogas in which a substrate comprising non-starch carbohydrate is contacted with a polypeptide or composition of the invention, thereby to yield fermentable material which may be converted into a biogas by an organism such as a microorganism.
- a polypeptide of the invention may be provided by way of an organism, for example a microorganism which expresses such a polypeptide.
- polypeptides and compositions of the invention may be used in a method of processing plant material to degrade or modify the cellulose or hemicellulose or pectic substance constituents of the cell walls of the plant or fungal material. Such methods may be useful in the preparation of food product. Accordingly, the invention provides a method for preparing a food product which method comprises incorporating a polypeptide or composition of the invention during preparation of the food product.
- the invention also provides a method of processing a plant material which method comprises contacting the plant material with a polypeptide or composition of the invention to degrade or modify the cellulose in the (plant) material.
- the plant material is a plant pulp or plant extract, such as juices.
- the present invention also provides a method for reducing the viscosity, clarity and/or filterability of a plant extract which method comprises contacting the plant extract with a polypeptide or composition of the invention in an amount effective in degrading cellulose or hemicellulose or pectic substances contained in the plant extract.
- Plant and cellulose/hemicellulose/pectic substance-containing materials include plant pulp, parts of plants and plant extracts.
- an extract from a plant material is any substance which can be derived from plant material by extraction (mechanical and/or chemical), processing or by other separation techniques.
- the extract may be juice, nectar, base, or concentrates made thereof.
- the plant material may comprise or be derived from vegetables, e.g., carrots, celery, onions, legumes or leguminous plants (soy, soybean, peas) or fruit, e.g., pome or seed fruit (apples, pears, quince etc.), grapes, tomatoes, citrus (orange, lemon, lime, mandarin), melons, prunes, cherries, black currants, redcurrants, raspberries, strawberries, cranberries, pineapple and other tropical fruits, trees and parts thereof (e.g. pollen, from pine trees), or cereal (oats, barley, wheat, maize, rice).
- the material (to be hydrolysed) may also be agricultural residues, such as sugar beet pulp, com cobs, wheat straw, (ground) nutshells, or recyclable materials, e.g. (waste) paper.
- polypeptides of the invention can thus be used to treat plant material including plant pulp and plant extracts. They may also be used to treat liquid or solid foodstuffs or edible foodstuff ingredients, or be used in the extraction of coffee, plant oils, starch or as a thickener in foods.
- the polypeptides of the invention are used as a composition/enzyme preparation as described above.
- the composition will generally be added to plant pulp obtainable by, for example mechanical processing such as crushing or milling plant material. Incubation of the composition with the plant will typically be carried out for at time of from 10 minutes to 5 hours, such as 30 minutes to 2 hours, preferably for about 1 hour.
- the processing temperature is preferably from about 10°C to about 55°C, e. g. from about 15°C to about 25°C, optimally about 20°C and one can use from about 10 g to about 300 g, preferably from about 30 g to about 70 g, optimally about 50 g of enzyme per ton of material to be treated.
- All of the enzyme(s) or their compositions used may be added sequentially or at the same time to the plant pulp.
- the plant material may first be macerated (e.g. to a pure) or liquefied.
- processing parameters such as the yield of the extraction, viscosity of the extract and/or quality of the extract can be improved.
- a polypeptide of the invention may be added to the raw juice obtained from pressing or liquefying the plant pulp. Treatment of the raw juice will be carried out in a similar manner to the plant pulp in respect of dosage, temperature and holding time. Again, other enzymes such as those discussed previously may be included. Typical incubation conditions are as described in the previous paragraph.
- the juice is then centrifuged or (ultra) filtered to produce the final product.
- the (end) product can be heat treated, e.g. at about 100°C for a time of from about 1 minute to about 1 hour, under conditions to partially or fully inactivate the polypeptide(s) of the invention.
- composition containing a polypeptide of the invention may also be used during the preparation of fruit or vegetable purees.
- the polypeptide of the invention may also be used in brewing, wine making, distilling or baking. It may therefore be used in the preparation of alcoholic beverages such as wine and beer. For example, it may improve the filterability or clarity, for example of beers, wort (e.g. containing barley and/or sorghum malt) or wine.
- a polypeptide or composition of the invention may be used for treatment of brewers spent grain, i.e. residuals from beer wort production containing barley or malted barley or other cereals, so as to improve the utilization of the residuals for, e.g., animal feed.
- the protein may assist in the removal of dissolved organic substances from broth or culture media, for example where distillery waste from organic origin is bioconverted into microbial biomass.
- the polypeptide of the invention may improve filterability and/or reduce viscosity in glucose syrups, such as from cereals produced by liquefaction (e.g. with ⁇ -amylase).
- the polypeptide may improve the dough structure, modify its stickiness or suppleness, improve the loaf volume and/or crumb structure or impart better textural characteristics such as break, shred or crumb quality.
- the present invention thus relates to methods for preparing a dough or a cereal-based food product comprising incorporating into the dough a polypeptide or composition of the present invention. This may improve one or more properties of the dough or the cereal-based food product obtained from the dough relative to a dough or a cereal-based food product in which the polypeptide is not incorporated.
- the preparation of the cereal-based food product according to the invention further can comprise steps known in the art such as boiling, drying, frying, steaming or baking of the obtained dough.
- Products that are made from a dough that is boiled are for example boiled noodles, dumplings, products that are made from fried dough are for example doughnuts, buttons, fried noodles, products that are made for steamed dough are for example steamed buns and steamed noodles, examples of products made from dried dough are pasta and dried noodles and examples of products made from baked dough are bread, cookies and cake.
- improved property is defined herein as any property of a dough and/or a product obtained from the dough, particularly a cereal-based food product, which is improved by the action of the polypeptide according to the invention relative to a dough or product in which the polypeptide according to the invention is not incorporated.
- the improved property may include, but is not limited to, increased strength of the dough, increased elasticity of the dough, increased stability of the dough, improved machinability of the dough, improved proofing resistance of the dough, reduced stickiness of the dough, improved extensibility of the dough, increased volume of the cereal-based food product, reduced blistering of the cereal-based food product, improved crumb structure of the baked product, improved softness of the cereal-based food product, improved flavour of the cereal-based food product, improved anti-staling of the cereal-based food product.
- Improved properties related to pasta and noodle type of cereal-based products are for example improved firmness, reduced stickiness, improved cohesiveness and reduced cooking loss.
- the improved property may be determined by comparison of a dough and/or a cereal-based food product prepared with and without addition of a polypeptide of the present invention.
- Organoleptic qualities may be evaluated using procedures well established in the baking industry, and may include, for example, the use of a panel of trained taste-testers.
- the term "dough” is defined herein as a mixture of cereal flour and other ingredients firm enough to knead or roll.
- cereals are wheat, rye, corn, maize, barley, rice, groats, buckwheat and oat.
- Wheat is I here and hereafter intended to encompass all known species of Triticum genus, for example aestivum, durum and/or spelt.
- suitable other ingredients are: the cellobiohydrolase according to the present invention, additional enzymes, chemical additives and/or processing aids.
- the dough may be fresh, frozen, pre-pared, or pre-baked.
- the preparation of a dough from the ingredients described above is well known in the art and comprises mixing of said ingredients and processing aids and one or more moulding and optionally fermentation steps.
- the preparation of frozen dough is described by Kulp and Lorenz in Frozen and Refrigerated Doughs and Batters.
- cereal-based food product is defined herein as any product prepared from a dough, either of a soft or a crisp character.
- cereal-based food products whether of a white, light or dark type, which may be advantageously produced by the present invention are bread (in particular white, whole-meal or rye bread), typically in the form of loaves or rolls, French baguette-type bread, pasta, noodles, doughnuts, bagels, cake, pita bread, tortillas, tacos, cakes, pancakes, biscuits, cookies, pie crusts, steamed bread, and crisp bread, and the like.
- baked product is defined herein as any cereal-based food product prepared by baking the dough.
- Non-starch polysaccharides can increase the viscosity of the digesta which can, in turn, decrease nutrient availability and animal performance.
- the use of the cellobiohydrolase of the present invention can improve phosphorus utilization as well as cation minerals and protein during animal digesta.
- Non-starch polysaccharides are also present in virtually all feed ingredients of plant origin. NSPs are poorly utilized and can, when solubilized, exert adverse effects on digestion. Exogenous enzymes can contribute to a better utilization of these NSPs and as a consequence reduce any anti-nutritional effects.
- Non-starch carbohydrate degrading enzymes of the present invention can be used for this purpose in cereal-based diets for poultry and, to a lesser extent, for pigs and other species.
- a non-starch carbohydrate degrading polypeptide/enzyme of the invention (of a composition comprising the polypeptide/enzyme of the invention) may be used in the detergent industry, for example for removal from laundry of carbohydrate-based stains.
- a detergent composition may comprise a polypeptide/enzyme of the invention and, in addition, one or more of a cellulase, a hemicellulase, a pectinase, a protease, a lipase, a cutinase, an amylase or a carbohydrase.
- a detergent composition comprising a polypeptide or composition of the invention may be in any convenient form, for example a paste, a gel, a powder or a liquid.
- a liquid detergent may be aqueous, typically containing up to about 70% water and from about 0 to about 30% organic solvent or non-aqueous material.
- Such a detergent composition may, for example, be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dish washing operations.
- the properties of the enzyme should be compatible with the aselected detergent (for example, pH-optimum, compatibility with other enzymatic and/or non-enzymatic ingredients, etc.) and the enzyme(s) should be present in an effective amount.
- the aselected detergent for example, pH-optimum, compatibility with other enzymatic and/or non-enzymatic ingredients, etc.
- a detergent composition may comprise a surfactant, for example an anionic or nonionic surfactant, a detergent builder or complexing agent, one or more polymers, a bleaching system (for example an H 2 O 2 source) or an enzyme stabilizer.
- a detergent composition may also comprise any other conventional detergent ingredient such as, for example, a conditioner including a clay, a foam booster, a sud suppressor, an anti-corrosion agent, a soil-suspending agent, an an-soil redeposition agent, a dye, a bactericide, an optical brightener, a hydrotropes, a tarnish inhibitor or a perfume.
- a polypeptide or composition of the present invention may be used in the paper and pulp industry, inter alia in the bleaching process to enhance the brightness of bleached pulps whereby the amount of chlorine used in the bleaching stages may be reduced, and to increase the freeness of pulps in the recycled paper process ( Eriksson, K. E. L., Wood Science and Technology 24 (1990):79-101 ; Paice, et al., Biotechnol. and Bioeng. 32 (1988):235-239 and Pommier et al., Tappi Journal (1989):187-191 ).
- a polypeptide or composition of the invention may be used for treatment of lignocellulosic pulp so as to improve the bleachability thereof. Thereby the amount of chlorine need to obtain a satisfactory bleaching of the pulp may be reduced.
- a polypeptide or composition of the invention may be used in a method of reducing the rate at which cellulose-containing fabrics become harsh or of reducing the harshness of cellulose-containing fabrics, the method comprising treating cellulose-containing fabrics with a polypeptide or composition as described above.
- the present invention further relates to a method providing colour clarification of coloured cellulose-containing fabrics, the method comprising treating coloured cellulose-containing fabrics with a polypeptide or composition as described above, and a method of providing a localized variation in colour of coloured cellulose-containing fabrics, the method comprising treating coloured cellulose-containing fabrics with a polypeptide or composition as described above.
- the methods of the invention may be carried out by treating cellulose-containing fabrics during washing. However, if desired, treatment of the fabrics may also be carried out during soaking or rinsing or simply by adding the polypeptide or composition as described above to water in which the fabrics are or will be immersed.
- a polypeptide or composition of the present invention can also be used in antibacterial formulation as well as in pharmaceutical products such as throat lozenges, toothpastes, and mouthwash.
- Aspergillus niger strain is deposited at the CBS Institute under the deposit number CBS 513.88.
- TEC-142 Rasamsonia (Talaromyces) emersonii strain TEC-142 is deposited at CENTRAAL BUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands on 1st July 2009 having the Accession Number CBS 124902.
- TEC-142S is a single isolate of TEC-142.
- Rasamsonia (Talaromyces) emersonii strain was deposited at CENTRAAL BUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands in December 1964 having the Accession Number CBS 393.64.
- Other suitable strains can be equally used in the present examples to show the effect and advantages of the invention.
- TEC-101, TEC-147, TEC-192, TEC-201 or TEC-210 are suitable Rasamsonia strains which are described in WO2011/000949 .
- TEC-210 cellulase-containing composition was produced according to the procedures such as inoculation and fermentation as described in WO2011/000949 .
- Beta-glucosidase is produced by overexpression of EBA4 in Aspergillus niger as described in WO2011/098577 followed by fermentation of the Aspergillus niger transformant.
- EBA4 is a Rasamsonia emersonii ( Talaromyces emersonii ) BG and is identified in WO2011/098577 as T. emersonii beta-glucosidase (BG) and represented by SEQ ID NO: 5 in WO2011/098577 .
- All gene replacement vectors comprise approximately 1 - 2 kb flanking regions of the respective ORF sequences, to target for homologous recombination at the predestined genomic loci.
- A. niger vectors contain the A. nidulans bi-directional amd S selection marker for transformation, in-between direct repeats.
- the method applied for gene deletion in all examples herein uses linear DNA, which integrates into the genome at the homologous locus of the flanking sequences by a double cross-over, thus substituting the gene to be deleted by the amd S gene.
- the direct repeats allow for the removal of the selection marker by a (second) homologous recombination event.
- amd S marker can be used indefinitely in strain modification programs.
- Potato dextrose agar, PDA (Fluka, Cat. No. 70139): per litre: Potato extrac 4 g; Dextrose 20 g; Bacto agar 15 g; pH 5.4; Sterilize 20 min at 120°C.
- Rasamsonia agar medium per litre: S alt fraction no.3 15 g; Cellulose 30 g; Bacto peptone 7.5 g; Grain flour 15 g; KH2PO4 5 g; CaCl2.2aq 1 g; Bacto agar 20 g; pH 6.0; Sterilize 20 min at 120°C.
- Salt fraction composition The "salt fraction no.3" was fitting the disclosure of WO98/37179 , Table 1. Deviations from the composition of this table were CaCl2.2aq 1.0 g/l, KCl 1.8 g/L, citric acid 1aq 0.45 g/L (chelating agent).
- Rasamsonia medium 1 per litre: Glucose 20 g; Yeast extract (Difco) 20 g; Clerol FBA3107 (AF) 4 drops; pH 6.0; Sterilize 20 min at 120°C.
- Rasamsonia medium 2 per litre: Salt fraction no.3 15 g; Cellulose 20 g; Bacto peptone 4 g; Grain flour 7.5 g; KH2PO4 10 g; CaCl2.2H20 0.5 g; Clerol FBA3107 (AF) 0.4 ml; pH 5; Sterilize 20 min at 120°C.
- Rasamsonia medium 3 per litre: Salt fraction no.3 15 g; glucose 50 g; Bacto peptone 7.5 g; KH2PO4 10 g; CaCl2.2H20 0.5 g; Clerol FBA3107 (AF) 0.4 ml; pH 5; Sterilize 20 min at 120°C.
- Strains were grown from stocks on Rasamsonia agar medium in 10 cm diameter Petri dishes for 5-7 days at 40°C. For MTP fermentations, strains were grown in 96-well plates containing Rasamsonia agar medium. Strain stocks were stored at -80°C in 10% glycerol.
- Strains were grown in YGG medium (per liter: 8 g KCI, 16 g glucose.H2O, 20 ml of 10% yeast extract, 10 ml of 100x pen/strep, 6.66 g YNB+amino acids, 1.5 g citric acid, and 6 g K2HPO4). for 16 hours at 42°C, 250 rpm, and chromosomal DNA was isolated using the DNeasy plant mini kit (Qiagen, Hilden, Germany).
- 96 wells microtiter plates with sporulated R. emersonii strains were used to harvest spores for MTP fermentations.
- 200 ⁇ l of 10 times diluted Rasamsonia medium 1 was added to each well and after resuspending the mixture, 100 ⁇ l of spore suspension was incubated in humidity shakers (Infors) for 44°C at 550 rpm, and 80% humidity for 16 hours.
- 50 ⁇ l of pre-culture was used to inoculate 250 ⁇ l of Rasamsonia medium 2 in MTP plates.
- the 96-well plates were incubated in humidity shakers (Infors) for 44°C at 550 rpm, and 80% humidity for 6 days. Plates were centrifuged and supernatants were harvested.
- Spores were directly inoculated into 500 ml shake flasks containing 100 ml of either Rasamsonia medium 2 or 3 and incubated at 45°C at 250 rpm in an incubator shaker for 3-4 days.
- spores were inoculated in 100 ml shake flasks containing Rasamsonia medium 1 and incubated at 45°C at 250 rpm in an incubator shaker for 1 day (preculture) and, subsequently, 5 or 10 ml of biomass from the pre-culture was transferred to 500 ml shake flasks containing 100 ml of Rasamsonia medium 2 or 3 and grown under conditions as described above.
- Protein samples were separated under reducing conditions on NuPAGE 4-12% Bis-Tris gel (Invitrogen, Breda, The Netherlands) and stained. Gels were stained with either InstantBlue (Expedeon, Cambridge, United Kingdom), SimplyBlue safestain (Invitrogen, Breda, The Netherlands) or Sypro Ruby (Invitrogen, Breda, The Netherlands) according to manufacturer's instructions.
- Protein content of the recovered supernatant was determined according to Bradford method. The amount of protein in the enzyme samples was determined with Bradford Protein assay, using Coomassie protein reagent. 25 ⁇ l of appropriately diluted enzyme sample was mixed with 1.2 ml Coomassie reagent. After 10 minutes at room temperature the absorbance of the mixture at 595 nm was determined using a spectrophotometer (Uvikon XL). Protein content was calculated in comparison to BSA standard.
- the enzyme culture supernatant was analysed in duplicate according to the following procedure: 5 mg protein /g dry matter feedstock of the enzyme culture supernatant was transferred to a suitable vial containing 800 ⁇ L 2.5 % ( w / w ) dry matter of a mildly acid pre-treated corn stover substrate in a 50 mM citrate buffer, buffered at pH 3.5 or pH 4.5 or 5.0.
- the enzyme culture supernatant was also tested in combination with two different hemicellulase mixtures; TEC-210 ( Rasamsonia emersonii ) to which additional beta-glucosidase (BG) ( Aspergillus niger strain expressing a BG from Rasamsonia emersonii ) was added (0.08 mg/g dry matter) and Celluclast ( Trichoderma reesei ) to which additional BG (Novozym-188) was added (0.08 mg/g dry matter). The mixtures were added to a concentration of 1 mg protein/ g dry matter of the feedstock. These incubations were performed at the same conditions as described above.
- the (hemi)-cellulase enzyme solution may contain residual sugars. Therefore, the results of the assay are corrected for the sugar content measured after incubation of the enzyme solution.
- This assay measures the release of p -nitrophenol by the action of ⁇ -xylosidase on p -nitrophenyl- ⁇ -D-xylopyranoside (PNPX).
- PNPX p -nitrophenyl- ⁇ -D-xylopyranoside
- One ⁇ -xylosidase unit of activity is the amount of enzyme that liberates 1 micromole of p -nitrophenol in one minute at 60°C and pH 4.5.
- Acetate buffer (0.1 M, pH 4.5) is prepared as follows: 8.2 g of anhydrous sodium acetate is dissolved in distilled water so that the final volume of the solution is 1000 ml (Solution A).
- PNPX 100 mg of PNPX is dissolved in 84 mL of 0.1 M acetate buffer to obtain a 4.4 mM stock solution.
- the stop reagent (1 M sodium carbonate solution) is prepared as follows: 10.6 g of anhydrous sodium carbonate is dissolved in 50 ml of distilled water, and the solution volume is adjusted to 100 ml. This reagent is used to terminate the enzymatic reaction.
- 0.1 mL of 4.4 mM PNPX stock solution is mixed with 0.1 mL of the appropriate diluted enzyme sample and incubated at 60°C for 60 minutes. After 60 minutes of incubation, 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solution and the absorbance is measured at 405 nm in microtiter plates as As
- 0.1 mL of 4.4 mM PNPX stock solution is mixed with 0.1 mL of 0.1 M acetate buffer, pH 4.5 and treated the same as the samples: incubated at 60°C for 60 minutes after which 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solution and the absorbance at 405 nm is measured in microtiter plates as A SB .
- Enzyme blanks (without addition of substrate) are measured to correct for background color originating from the enzymes.
- 0.1 mL of the appropriate diluted enzyme sample is mixed with 0.1 mL 0.1 M acetate buffer, pH 4.5 and incubated at 60°C for 60 minutes. After 60 minutes of incubation, 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solution and the absorbance is measured at 405 nm in microtiter plates as A EB .
- a calibration curve of p -nitrophenol (appropriate diluted in 0.1 M acetate buffer, pH 4.5) mixed in a ratio of 1:1 with 1 M sodium carbonate solution is used to quantify its release from PNPX by the action of the enzyme.
- the activity is expressed as the amount of enzyme required to release 1 ⁇ M p- nitrophenol /min under the assay conditions.
- This assay can be used to test the activity of enzymes such as, but not limited to, GH3, GH30, GH39, GH43, GH52, and GH54 enzymes.
- This assay measures the release of xylose by the action of ⁇ -xylosidase on xylobiose.
- Sodium acetate buffer (0.05 M, pH 4.5) was prepared as follows. 4.1 g of anhydrous sodium acetate or 6.8 g of sodium acetate * 3H 2 O was dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) of glacial acetic acid was mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer, pH 4.5, was prepared by mixing Solution A with Solution B until the pH of the resulting solution was equal to 4.5. Xylobiose was purchased from Sigma and dissolved in sodium acetate buffer pH 4.5 to a concentration of 100 ug/mL
- the assay was performed as detailed below.
- the enzyme culture supernatant was added to the substrate in a dosage of 1 and 5 mg protein/ g substrate which was then incubated at 62°C for 24 hours. The reaction was stopped by heating the samples for 10 minutes at 100°C. The release of xylose was analyzed by High Performance Anion Exchange Chromatography.
- a flow rate of 0.3 mL/min was used with the following gradient of sodium acetate in 0.1 M NaOH: 0-20 min, 0-180 mM. Each elution was followed by a washing step of 5 min 1000 mM sodium acetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 M NaOH.
- This assay can be used to test the activity of enzymes such as, but not limited to, GH3, GH30, GH39, GH43, GH52, and GH54 enzymes.
- xylan substrates like Oat arabinoxylan, Beech wood xylan and Birch wood xylan (Sigma) instead of xylobiose to measure xylosidase activity on polymeric substrates.
- Assay conditions were the same with the exception that all substrates were solved to a concentration of 2 mg/mL.
- the incubation was performed at 60 °C for 24h at a dosage of 10 mg/g.
- This assay measures the release of p-nitrophenol by the action of ⁇ -galactosidase on p-nitrophenyl- ⁇ -D-galactopyranoside (PNPG).
- PNPG p-nitrophenyl- ⁇ -D-galactopyranoside
- One ⁇ -galactosidase unit of activity is the amount of enzyme that liberates 1 micromole of p-nitrophenol in one minute at 60°C and pH 4.5.
- Acetate buffer (0.1 M, pH 4.5) is prepared as follows: 8.2 g of anhydrous sodium acetate is dissolved in distilled water so that the final volume of the solution is 1000 ml (Solution A).
- a stock solution of 4.4 mM PNPG is made in 0.1 M acetate buffer.
- the stop reagent (1 M sodium carbonate solution) is prepared as follows: 10.6 g of anhydrous sodium carbonate is dissolved in 50 ml of distilled water, and the solution volume is adjusted to 100 ml. This reagent is used to terminate the enzymatic reaction.
- 0.1 mL of 4.4 mM PNPG stock solution is mixed with 0.1 mL of the appropriate diluted enzyme sample and incubated at 60°C for 60 minutes. After 60 minutes of incubation, 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solution and the absorbance is measured at 405 nm in microtiter plates as As
- 0.1 mL of 4.4 mM PNPG stock solution is mixed with 0.1 mL of 0.1 M acetate buffer, pH 4.5 and treated the same as the samples: incubated at 60°C for 60 minutes after which 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solution and the absorbance at 405 nm is measured in microtiter plates as A SB .
- Enzyme blanks (without addition of substrate) are measured to correct for background color originating from the enzymes.
- 0.1 mL of the appropriate diluted enzyme sample is mixed with 0.1 mL 0.1 M acetate buffer, pH 4.5 and incubated at 60°C for 60 minutes. After 60 minutes of incubation, 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solution and the absorbance is measured at 405 nm in microtiter plates as A EB .
- a calibration curve of p -nitrophenol (appropriate diluted in 0.1 M acetate buffer, pH 4.5) mixed in a ratio of 1:1 with 1 M sodium carbonate solution is used to quantify its release from PNPG by the action of the enzyme.
- the activity is expressed as the amount of enzyme required to release 1 ⁇ M p- nitrophenol /min under the assay conditions.
- This assay can be used to test the activity of enzymes such as, but not limited to, GH4, GH27 and GH36 enzymes.
- Sodium acetate buffer (0.05 M, pH 4.5) was prepared as follows. 4.1 g of anhydrous sodium acetate was dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) of glacial acetic acid was mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer, pH 4.5, was prepared by mixing Solution A with Solution B until the pH of the resulting solution is 4.5.
- Tamarind xyloglucan was solved in sodium acetate buffer to obtain 2.0 mg/mL.
- the enzyme culture supernatant was added to the substrate in a dosage of 10 mg protein/ g substrate which was then incubated at 60°C for 24 hours. The reaction was stopped by heating the samples for 10 minutes at 100°C.
- the release of oligosaccharides was analyzed by High Performance Anion Exchange Chromatography As a blank sample the substrate was treated and incubated in the same way but then without the addition of enzyme.
- the substrate was also incubated under the same conditions with a commercial cellulase preparation from Trichoderma Reesei (Celluclast; Sigma) which was diluted 50 times after which 20 ⁇ L was added to the incubation.
- the analysis was performed using a Dionex HPLC system equipped with a Dionex CarboPac PA-1 (2 mm ID x 250 mm) column in combination with a CarboPac PA guard column (2 mm ID x 50 mm) and a Dionex PAD-detector (Dionex Co. Sunnyvale).
- a flow rate of 0.3 mL/min was used with the following gradient of sodium acetate in 0.1 M NaOH: 0-40 min, 0-150 mM.
- Each elution was followed by a washing step of 5 min 1000 mM sodium acetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 M NaOH.
- This assay can be used to test the activity of enzymes such as, but not limited to, GH5, GH12, GH16, GH44, and GH74 enzymes.
- the following example illustrates the assay to measure xyloglucanase activity. Such activity was demonstrated by using xyloglucan as substrate and a reducing sugars assay (PAHBAH) as detection method. The values were compared to a standard, which was prepared using a commercial cellulase preparation from Trichoderma Reesei (Celluclast; Sigma).
- Reagent A 5 g of p-Hydroxybenzoic acid hydrazide (PAHBAH) was suspended in 60 mL water, 4.1 mL of concentrated hydrochloric acid was added and the volume was adjusted to 100 ml.
- Reagent B 24.9 g of trisodium citrate was dissolved in 500 ml of water. To this solution 2.2 g of calcium chloride and 40 g sodium hydroxide was added. The volume was adjusted to 2 L with water. Both reagents were stored at room temperature.
- Working Reagent 10 ml of Reagent A was added to 40 ml of Reagent B. This solution was prepared freshly every day, and was stored on ice between uses. Using the above reagents, the assay was performed as detailed below
- the substrates were also incubated without addition of enzyme culture supernatant and the enzyme culture supernatants were incubated without substrate.
- This assay can be used to test the activity of enzymes such as, but not limited to, GH5, GH12, GH16, GH44, and GH74 enzymes.
- Sodium acetate buffer (0.05 M, pH 4.5) is prepared as follows. 4.1 g of anhydrous sodium acetate is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer, pH 4.5, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is 4.5.
- Tamarind xyloglucanan is solved in sodium acetate buffer to obtain 2.0 mg/mL.
- the enzyme is added to the substrate in a dosage of 10 mg protein/ g substrate which is then incubated at 60°C for 24 hours. The reaction is stopped by heating the samples for 10 minutes at 100°C.
- the formation of lower molecular weight oligosaccharides is analyzed by High Performance size-exclusion Chromatography As a blank sample the substrate is treated and incubated in the same way but then without the addition of enzyme.
- the substrate is also incubated under the same conditions with a commercial cellulase preparation from e.g. Aspergillus niger or Trichoderma Reesei (the cellulase standard at its own optimal temperature in case of inactivity at 60°C).
- a commercial cellulase preparation from e.g. Aspergillus niger or Trichoderma Reesei (the cellulase standard at its own optimal temperature in case of inactivity at 60°C).
- HPSEC High-performance size-exclusion chromatography
- TSK-gel columns 6.0 mm ⁇ 15.0 cm per column
- SuperAW4000 SuperAW3000
- SuperAW2500 Tosoh Bioscience
- PWXguard column Tosoh Bioscience
- Elution is performed at 55 C with 0.2 M sodium nitrate at 0.6 mL/min.
- the eluate was monitored using a Shodex RI-101 (Kawasaki) refractive index (RI) detector.
- Calibration was performed by using pullulans (Associated Polymer Labs Inc., New York, USA) with a molecular weight in the range of 0.18-788 kDa.
- This assay can be used to test the activity of enzymes such as, but not limited to, GH5, GH12, GH16, GH44, and GH74 enzymes.
- the following example illustrates an assay to measure the ability of ⁇ -arabinofuranosidases to remove the ⁇ -L-arabinofuranosyl residues from substituted xylose residues.
- arabinoxylans For the complete degradation of arabinoxylans to arabinose and xylose, several enzyme activities are needed, including endo-xylanases and arabinofuranosidases.
- the arabinoxylan molecule from wheat is highly substituted with arabinosyl residues. These can be substituted either to the C2 or the C3 position of the xylosyl residue (single substitution), or both to the C2 and C3 position of the xylose (double substitution).
- Single and double substituted oligosaccharides were prepared by incubating wheat arabinoxylan (WAX; 10 mg/mL; Megazyme, Bray, Ireland) in 50 mM acetate buffer pH 4,5 with an appropriate amount of endo-xylanase (from Aspergillus awamori, Kormelink F. et al; Journal of Biotechnology (1993) 27: 249-265 ) 48 hours at 40°C to produce an sufficient amount of arabinoxylo-oligosaccharides. The reaction was stopped by heating the samples at 100°C for 10 minutes. The samples were centrifuged for 5 minutes at 10.000 x g. The supernatant was used for further experiments. Degradation of the arabinoxylan was followed by analysis of the formed reducing sugars and High Performance Anion Exchange Chromatography (HPAEC).
- HPAEC High Performance Anion Exchange Chromatography
- the enzyme culture supernatant was added to the single and double substituted arabinoxylo-oligosaccharides (endo-xylanase treated WAX; 2 mg/mL) in a dosage of 10 mg protein/ g substrate in 50 mM sodium acetate buffer which was then incubated at 65°C for 24 hours. The reaction was stopped by heating the samples at 100°C for 10 minutes. The samples were centrifuged for 5 minutes at 10.000 x g. The release of arabinose was followed by HPAEC analysis.
- the analysis was performed using a Dionex HPLC system equipped with a Dionex CarboPac PA-1 (2 mm ID x 250 mm) column in combination with a CarboPac PA guard column (2 mm ID x 50 mm) and a Dionex PAD-detector (Dionex Co. Sunnyvale).
- a flow rate of 0.3 mL/min was used with the following gradient of sodium acetate in 0.1 M NaOH: 0-40 min, 0-400 mM.
- Each elution was followed by a washing step of 5 min 1000 mM sodium acetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 M NaOH.
- Arabinose release was identified and quantified by a standard (Sigma).
- This assay can be used to test the activity of enzymes such as, but not limited to, GH3, GH43, GH51, GH54, and GH62 enzymes.
- Endo-xylanases are enzyme able to hydrolyze ⁇ -1,4 bond in the xylan backbone, producing short xylooligosaccharides.
- This assay measures the release of xylose and xylo-oligosaccharides by the action of xylanases on wheat arabinoxylan (WAX) (Megazyme, Medium viscosity 29 cSt), Oat arabinoxylan, Beech wood xylan and Birch wood xylan (Sigma).
- Sodium acetate buffer (0.05 M, pH 4.5) was prepared as follows; 4.1 g of anhydrous sodium acetate was dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) of glacial acetic acid was mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer, pH 4.5, was prepared by mixing Solution A with Solution B until the pH of the resulting solution was 4.5. Each substrate was solved in sodium acetate buffer to obtain 2.0 mg/mL.
- the enzyme culture supernatant was added to the substrate in a dosage of 10 mg protein/ g substrate which was then incubated at 60°C for 20 hours. The reaction was stopped by heating the samples for 10 minutes at 100°C. The release of xylose and xylooligosaccharides was analyzed by High Performance Anion Exchange Chromatography.
- the analysis was performed using a Dionex HPLC system equipped with a Dionex CarboPac PA-1 (2 mm ID x 250 mm) column in combination with a CarboPac PA guard column (2 mm ID x 50 mm) and a Dionex PAD-detector (Dionex Co. Sunnyvale).
- a flow rate of 0.3 mL/min was used with the following gradient of sodium acetate in 0.1 M NaOH: 0-40 min, 0-400 mM.
- Each elution was followed by a washing step of 5 min 1000 mM sodium acetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 M NaOH.
- Standards of xylose, xylobiose and xylotriose (Sigma) were used to identify these oligomers released by the action of the enzyme.
- This assay can be used to test the activity of enzymes such as, but not limited to, GH5, GH8, GH10, and GH11.
- This assay measures the release of p -nitrophenol by the action of ⁇ / ⁇ -xylosidase on p -nitrophenyl- ⁇ / ⁇ -D-xylopyranoside (PNPX).
- One ⁇ -xylosidase unit of activity is the amount of enzyme that liberates 1 micromole of p -nitrophenol in one minute at 60°C and pH 4.5.
- Acetate buffer (0.1 M, pH 4.5) is prepared as follows: 8.2 g of anhydrous sodium acetate is dissolved in distilled water so that the final volume of the solution is 1000 ml (Solution A).
- PNPX 100 mg of PNPX is dissolved in 84 mL of 0.1 M acetate buffer to obtain a 4.4 mM stock solution.
- the stop reagent (1 M sodium carbonate solution) is prepared as follows: 10.6 g of anhydrous sodium carbonate is dissolved in 50 ml of distilled water, and the solution volume is adjusted to 100 ml. This reagent is used to terminate the enzymatic reaction.
- 0.1 mL of 4.4 mM PNPX stock solution is mixed with 0.1 mL of the appropriate diluted enzyme sample and incubated at 60°C for 60 minutes. After 60 minutes of incubation, 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solution and the absorbance is measured at 405 nm in microtiter plates as As
- 0.1 mL of 4.4 mM PNPX stock solution is mixed with 0.1 mL of 0.1 M acetate buffer, pH 4.5 and treated the same as the samples: incubated at 60°C for 60 minutes after which 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solution and the absorbance at 405 nm is measured in microtiter plates as A SB .
- Enzyme blanks (without addition of substrate) are measured to correct for background color originating from the enzymes.
- 0.1 mL of the appropriate diluted enzyme sample is mixed with 0.1 mL 0.1 M acetate buffer, pH 4.5 and incubated at 60°C for 60 minutes. After 60 minutes of incubation, 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solution and the absorbance is measured at 405 nm in microtiter plates as A EB .
- a calibration curve of p -nitrophenol (appropriate diluted in 0.1 M acetate buffer, pH 4.5) mixed in a ratio of 1:1 with 1 M sodium carbonate solution is used to quantify its release from PNPX by the action of the enzyme.
- the activity is expressed as the amount of enzyme required to release 1 ⁇ M p- nitrophenol /min under the assay conditions.
- This assay can be used to test the activity of enzymes such as, but not limited to, GH3, GH30, GH31, GH39, GH43, GH52, and GH54 enzymes.
- This assay measures the release of p -nitrophenol by the action of ⁇ / ⁇ -mannosidase on p -nitrophenyl- ⁇ / ⁇ -D-mannopyranoside (PNPM).
- PNPM p -nitrophenyl- ⁇ / ⁇ -D-mannopyranoside
- One ⁇ / ⁇ -mannosidase unit of activity is the amount of enzyme that liberates 1 micromole of p -nitrophenol in one minute at 60°C and pH 4.5.
- Acetate buffer (0.1 M, pH 4.5) is prepared as follows: 8.2 g of anhydrous sodium acetate is dissolved in distilled water so that the final volume of the solution is 1000 ml (Solution A).
- a stock solution of 4.4 mM PNPM is made in 0.1 M acetate buffer.
- the stop reagent (1 M sodium carbonate solution) is prepared as follows: 10.6 g of anhydrous sodium carbonate is dissolved in 50 ml of distilled water, and the solution volume is adjusted to 100 ml. This reagent is used to terminate the enzymatic reaction.
- 0.1 mL of 4.4 mM PNPM stock solution is mixed with 0.1 mL of the appropriate diluted enzyme sample and incubated at 60°C for 60 minutes. After 60 minutes of incubation, 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solution and the absorbance is measured at 405 nm in microtiter plates as As
- 0.1 mL of 4.4 mM PNPM stock solution is mixed with 0.1 mL of 0.1 M acetate buffer, pH 4.5 and treated the same as the samples: incubated at 60°C for 60 minutes after which 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solution and the absorbance at 405 nm is measured in microtiter plates as A SB .
- Enzyme blanks (without addition of substrate) are measured to correct for background color originating from the enzymes.
- 0.1 mL of the appropriate diluted enzyme sample is mixed with 0.1 mL 0.1 M acetate buffer, pH 4.5 and incubated at 60°C for 60 minutes. After 60 minutes of incubation, 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solution and the absorbance is measured at 405 nm in microtiter plates as A EB .
- a calibration curve of p -nitrophenol (appropriate diluted in 0.1 M acetate buffer, pH 4.5) mixed in a ratio of 1:1 with 1 M sodium carbonate solution is used to quantify its release from PNPM by the action of the enzyme.
- the activity is expressed as the amount of enzyme required to release 1 ⁇ M p- nitrophenol /min under the assay conditions.
- This assay can be used to test the activity of enzymes such as, but not limited to, GH1, GH2, GH5, GH38, GH47, GH92, and GH125 enzymes.
- Synthetic substrates Methyl caffeate, methyl coumarate, methyl sinapinate and methyl ferulate are obtained from Apin Chemicals. Activity towards these synthetic substrates is determined by incubating the enzyme with the substrate at a dosage of about 5 mg/g DM at a pH of 5.0 (50 mM sodium acetate buffer). The reaction will be done at 60°C for up to 24 h.
- the mobile phase is composed of (A) H2O + 1% (v/v) acetonitrile + 0.2% (v/v) acetic acid and (B) acetonitrile + 0.2% (v/v) acetic acid.
- the flow rate is 0.4 mL/min, and the column temperature is 30°C.
- the elution profile is as follows: first 5 min, isocratic 0%B; 5-23 min, linear from 0 to 50% B; 23-24 min, linear from 50 to 100% B; 24-27min, isocratic at 100%B; 27-28 min, linear from 100 to 0% B, followed by reconditioning of the column for 7 min.
- Spectral data are collected from 200 to 600 nm, and quantification is performed at 320 nm. Ferulic, caffeic, sinapic and coumaric acid contents are identified and quantified on the basis of standards.
- MS data are collected in the negative mode with an ion spray voltage of 3.5 kV, a capillary voltage of -20 V, and a capillary temperature of 350 C. Full MS scans are made within the range m/z 150-1500, and MS2 data of the most intense ions is obtained.
- This assay can be used to test the activity of enzymes such as, but not limited to, CE1 enzymes.
- Natural occurring substrate Arabinoxylan oligomers purified from pretreated corn fibre (CF) (1 mg/ml each) (Appeldoorn et al 2010) are incubated with ferulic acid esterases at a dosage of about 5 mg/g DM at a pH of 5.0 (50 mM sodium acetate buffer). The reaction will be done at 60°C for up to 24 h.
- the mobile phase is composed of (A) H2O + 1% (v/v) acetonitrile + 0.2% (v/v) acetic acid and (B) acetonitrile + 0.2% (v/v) acetic acid.
- the flow rate is 0.4 mL/min, and the column temperature is 30°C.
- the elution profile is as follows: first 5 min, isocratic 0%B; 5-23 min, linear from 0 to 50% B; 23-24 min, linear from 50 to 100% B; 24-27min, isocratic at 100%B; 27-28 min, linear from 100 to 0% B, followed by reconditioning of the column for 7 min.
- Spectral data are collected from 200 to 600 nm, and quantification is performed at 320 nm. Ferulic and coumaric acid contents are identified and quantified on the basis of standards.
- MS data are collected in the negative mode with an ion spray voltage of 3.5 kV, a capillary voltage of -20 V, and a capillary temperature of 350 C. Full MS scans are made within the range m/z 150-1500, and MS2 data of the most intense ions is obtained.
- the total amount of ester-linked ferulic acid in corn oligomers was determined after alkaline hydrolysis and ethylether extraction using the UHPLC method described above.
- This assay can be used to test the activity of enzymes such as, but not limited to, CE1 enzymes.
- the following example illustrates the assay to measure the ⁇ -glucuronidase activity towards aldouronic acids(megazyme). This assay measures the release of xylose and xylooligomers by the action of the ⁇ -glucuronidase on the glucuronoxylan oligosaccharides.
- Sodium acetate buffer (0.05 M, pH 4.5) was prepared as follows. 4.1 g of anhydrous sodium acetate was dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) of glacial acetic acid was mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer, pH 4.5, was prepared by mixing Solution A with Solution B until the pH of the resulting solution was 4.5.
- the aldouronic acids are solved in sodium acetate buffer to obtain 1.0 mg/mL.
- the enzyme culture supernatant was added to the substrate in a dosage of 1 and 10 mg protein/ g substrate which was then incubated at 60°C for 24 hours. The reaction was stopped by heating the samples for 10 minutes at 100°C.
- the release of xylooligomers as a result of the removal of 4-O-methyl glucuronic acid were analyzed by High Performance Anion Exchange Chromatography
- the analysis was performed using a Dionex HPLC system equipped with a Dionex CarboPac PA-1 (2 mm ID x 250 mm) column in combination with a CarboPac PA guard column (2 mm ID x 50 mm) and a Dionex PAD-detector (Dionex Co. Sunnyvale).
- a flow rate of 0.3 mL/min was used with the following gradient of sodium acetate in 0.1 M NaOH: 0-40 min, 0-400 mM.
- Each elution was followed by a washing step of 5 min 1000 mM sodium acetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 M NaOH.
- This assay can be used to test the activity of enzymes such as, but not limited to, GH67 and GH115 enzymes.
- This Example describes the construction of an expression construct for overexpression of Temer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 in A. niger.
- Genomic DNA of Rasamsonia emersonii strain CBS393.64 was sequenced and analysed. The gene with translated protein annotated as activity according in Table 1 was identified. Sequences of the R.
- emersonii Temer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 and Temer07305 gene comprising the codon-pair optimised ORF sequence, protein sequence, signal sequence, genomic sequence and wild-type cDNA sequence are shown in sequence listings SEQ ID NO: 1 to 75.
- SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66 or 71 is cloned into the pGBTOP vector ( Fig. 1 ) using Eco RI and Pac I sites, comprising the glucoamylase promoter and terminator sequence.
- the E.coli part was removed by Not I digestion prior to transformation of A. niger CBS 513.88.
- A. niger strain CBS513.88 is co-transformed with the expression constructs and an appropriate selection marker ( amd S or phleomycin) containing plasmid according to method described in the experimental information section.
- an appropriate selection marker amd S or phleomycin
- a large batch of spores is generated by plating spores or mycelia onto PDA plates (Potato Dextrose Agar, Oxoid), prepared according to manufacturer's instructions. After growth for 3-7 days at 30 degrees Celsius, spores are collected after adding 0.01% Triton X-100 to the plates.
- Example 2 Construction of a R. emersonii expression vectors.
- This Example describes the construction of an expression construct for overexpression Temer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 in R. emersonii.
- the expression cassette was targeted integrated into the RePepA locus.
- Two vectors were constructed according to routine cloning procedures for targeting into the RePepA locus.
- the insert fragments of both vectors together can be applied in the so-called "bipartite gene-targeting" method (Nielsen et al., 2006, 43: 54-64).
- This method is using two non-functional DNA fragments of a selection marker which are overlapping (see also WO2008113847 for further details of the bipartite method) together with gene-targeting sequences.
- the selection marker becomes functional by integration at a homologous target locus.
- the first vector Te pep.bbn (General layout as in Fig. 2 ) comprises a 1500 bp 5' flanking region approximately 1.5 kb upstream of the RePepA ORF for targeting in the RePepA locus (ORF and approximately 1500 bp of the RePepA promoter), a lox66 site, and the non-functional 5' part of the ble coding region driven by the A.
- nidulans gpdA promoter PgpdA-ble sequence missing the last 104 bases of the coding sequence at the 3' end of ble, SEQ ID NO: 79.
- a ccdB gene was inserted in between the 5' RePepA flanking region and the lox66 site.
- the second pEBA1006 vector (General layout as in Fig. 3 ) comprises the non-functional 3' part of the ble coding region and the A.
- nidulans trpC terminator (ble-TtrpC sequence missing the first 12 bases of the coding sequence at the 5' end of ble, SEQ ID NO: 80), a lox71 site, and a 2500 bp 3' flanking region of the RePepA ORF for targeting in the RePepA locus.
- the first and second non-functional fragments become functional producing a functional ble cassette.
- Both RePepA upstream and downstream gene flanking regions target for homologous recombination of the bipartite fragments at the predestined RePepA genomic locus.
- R. emersonii promoter 2 represented by SEQ ID NO: 81, is cloned upstream of the R.
- A. nidulans amdS terminator generating construct pEBA.
- the A. nidulans amdS terminator sequence is represented by SEQ ID NO: 82.
- a schematic representation of pEBA for overexpression of the Gene of interest (GOI) being Temer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 is shown in Figure 4 .
- Example 3 Overexpression of Temer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 gene in Rasamsonia emersonii
- Linear DNA of pEBA and pEBA1006 are isolated and used to transform Rasamsonia emersonii using method as described earlier in WO2011/054899 .
- the linear DNAs can integrate together into the genome at the RePepA locus, thus substituting the RePepA gene by the Temer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 and ble gene.
- Transformants are selected on phleomycin media and colony purified and tested according to procedures as described in WO2011/054899 .
- Growing colonies are diagnosed by PCR for integration at the RePepA locus using a primer in the gpdA promoter of the deletion cassette and a primer directed against the genomic sequence directly upstream of the 5' targeting region.
- Candidate transformants in which RePepA is replaced by Temer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 / ble cassettes are obtained.
- Example 4 enzymatic activity in Temer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 overexpressing Rasamsonia emersonii strains
- Temer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 overexpressing strains are fermented in shake flask in Rasamsonia medium 3 and supernatants are analysed for activity according to Table 1 in a suitable assay.
- Example 5 Aspergillus niger shake flask fermentation
- the fermentation supernatants obtained as described above were concentrated using a 10 kDa spin filter to a volume of approximately 5 ml. Subsequently, the protein concentration in the concentrated supernatant was determined via a TCA-biuret method.
- Concentrated protein samples were diluted with water to a concentration between 2 and 8 mg/ml.
- Bovine serum albumin (BSA) dilutions (0, 1, 2, 5, 8 and 10 mg/ml) were made and included as samples to generate a calibration curve.
- BSA bovine serum albumin
- 270 ⁇ l was transferred into a 10 ml tube containing 830 ⁇ l of a 12% (w/v) trichloro acetic acid solution in acetone and mixed thoroughly. Subsequently, the tubes were incubated on ice water for one hour and centrifuged for 30 minutes, at 4°C and 6000 rpm.
- the supernatant was discarded and pellets were dried by inverting the tubes on a tissue and letting them stand for 30 minutes at room temperature.
- 3 ml BioQuant Biuret reagent mix was added to the pellet in the tube and the pellet was solubilized upon mixing followed by addition of 1 ml water.
- the tube was mixed thoroughly and incubated at room temperature for 30 minutes. The absorption of the mixture was measured at 546 nm with a water sample used as a blank measurement and the protein concentration was calculated via the BSA calibration line.
- the beta-xylosidase activity of Rasamsonia emersonii Temer09484 was analysed as described above.
- the supernatant of the Temer09484 A. niger shake flask fermentation was concentrated and assayed in two dosages for xylose release from xylobiose after incubation for 24 hours at pH 4,5 and 62°C.
- the enzyme showed significant xylose release from xylobiose as shown in Table 3. This shows that Temer09484 has beta-xylosidase activity.
- Table 4 Effect of Rasamsonia emersonii Temer09484 on release of xylose from several xylan substrates after incubation for 24h at pH 4.5 and 60 °C at a dosage of 10 mg/g DM.
- ug/mL Substrate (2 mg/mL) xylose Beech wood xylan 320 Birch wood xylan 250 Oat arabinoxylan 334 * All substrates contain ⁇ 3.0 ug/mL xylose when no enzyme was added
- Example 8 Improvement of two different cellulose mixtures by addition of Temer09484 for the hydrolysis of lignocellulosic feedstocks
- Temer09484 A. niger shake flask fermentation was concentrated and spiked on a mild acid pretreated corn stover feedstock as described above.
- the enzyme showed significant xylose release from this feedstock in a wide range of temperatures (50, 65 and 75 °C) and pH values (3.5 - 4.5 - 5.0) used during the 72 hours of incubation as shown in Table 5. This shows that Temer09484 is important for the hydrolysis of lignocellulosic feedstocks.
- Temer09484 A. niger shake flask fermentation was also tested in combination with 2 different cellulose mixtures: TEC-210 and Celluclast, both with additional BG added.
- the xylose release from mildly acid pretreated corn stover was improved for both cellulose mixes by the addition of Temer09484 in a wide range of temperatures (50, 65 and 75 °C) and pH values (3.5 - 4.5 - 5.0) used during the 72 hours of incubation as shown in Table 6. This shows that Temer09484 can be used to improve cellulose mixes in a wide range of temperatures and pH values used for the hydrolysis of lignocellulosic feedstocks.
- Table 6 Effect of Rasamsonia emersonii Temer09484 when spiked to two different cellulose mixes on release of xylose (g/L) from mildly acid pretreated corn stover feedstock after 72h incubation at different temperature/pH conditions.
- the beta-xylosidase activity of Rasamsonia emersonii Temer00088 was analysed as described above.
- the supernatant of the Temer00088 A. niger shake flask fermentation was concentrated and assayed in two dosages for xylose release from xylobiose after incubation for 24 hours at pH 4,5 and 62°C.
- the enzyme showed significant xylose release from xylobiose as shown in Table 7. This shows that Temer00088 has beta-xylosidase activity.
- Table 8 Effect of Rasamsonia emersonii Temer00088 on release of xylose from several xylan substrates after incubation for 20h at pH 4.5 and 60 °C at a dosage of 10 mg/g DM. ug/mL* Substrate (2 mg/mL) xylose Beech wood xylan 373 Birch wood xylan 469 Oat arabinoxylan 298 * All substrates contain ⁇ 3.0 ug/mL xylose when no enzyme was added
- Example 11 Improvement of two different cellulose mixtures by addition of Temer00088 for the hydrolysis of lignocellulosic feedstocks
- Temer00088 A. niger shake flask fermentation was also tested in combination with 2 different cellulose mixtures: TEC-210 and Celluclast, both with additional BG added.
- the xylose release from mildly acid pretreated corn stover was improved for both cellulose mixes by the addition of Temer00088 in a wide range of temperatures (50, 65 and 75 °C) and pH values (3.5 - 4.5 - 5.0) used during the 72 hours of incubation as shown in Table 10. This shows that Temer00088 can be used to improve cellulose mixes in a wide range of temperatures and pH values used for the hydrolysis of lignocellulosic feedstocks.
- the xyloglucanase activity of Rasamsonia emersonii Temer04790 was analysed as described above.
- the supernatant of the Temer04790 A. niger shake flask fermentation was concentrated, added to the substrate xyloglucan and incubated for 24 hours at pH 4,5 and 60°C.
- the enzyme was able to release several oligomers as shown in Figure 6. This shows that Temer04790 is active on xyloglucan and releases similar oligomers as the commercial cellulase mix Celluclast from Trichoderma reesei.
- Temer04790 is specific towards xyloglucan as hardly any activity on CMC was seen in contrast to the cellulase mixture (Table 11). Furthermore, Temer04790 was still active at 75°C while the cellulase mixture was almost inactive on xyloglucan at 75°C.
- Table 11 Effect of Rasamsonia emersonii Temer04790 on the hydrolysis of xyloglucan (tamarind) and carboxymethylcellulose (CMC) (Sigma) measured by the formation of reducing ends expressed as glucose equivalents (ug/mL) after 24h incubation at pH 4.5 at 60 °C and 75°C. 60°C pH 4.5 75°C pH 4.5 xyloglucan CMC xyloglucan CMC no enzyme -17 -18 -18 -18 Temer04790 92 8 69 -9 cellulase mix* 64 132 5 47 * Celluclast from Thrichoderma reesei (Sigma)
- the arabinofuranosidase activity of Rasamsonia emersonii Temer05249 was analysed as described above.
- the supernatant of the Temer05249 A. niger shake flask fermentation was concentrated and added to arabinoxylooligomers at 10 mg/g followed by incubation for 24 hours at pH 4,5 and 65°C.
- the enzyme showed significant arabinose release from arabinoxylooligomers as shown in Table 12. This shows that Temer05249 has arabinofuranosidase activity.
- Table 12 Effect of Rasamsonia emersonii Temer05249 on the release of arabinose from wheat arabinoxylan, which was pre-incubated with an endo-xylanase, after incubation for 24h at pH 4.5 and 65 °C at a dosage of 10 mg/g DM.
- the endo-xylanase activity of Rasamsonia emersonii Temer03124 was analysed as described above.
- the supernatant of the Temer03124 A. niger shake flask fermentation was concentrated and added to several xylan substrates at 10 mg/g followed by incubation for 20 hours at pH 4,5 and 60°C.
- the enzyme showed significant release of xylose and a range of xylooligomers as shown in Table 13. This shows that Temer03124 has endo-xylanase activity.
- Table 13 Effect of Rasamsonia emersonii Temer03124 on release of xylose and xylose oligomers from several xylan substrates after incubation for 20h at pH 4.5 and 60 °C at a dosage of 10 mg/g DM.
- Temer08570 The endo-xylanase activity of Rasamsonia emersonii Temer08570 was analysed as described above. The supernatant of the Temer08570 A. niger shake flask fermentation was concentrated and added to several xylan substrates at 10 mg/g followed by incubation for 20 hours at pH 4,5 and 60°C. The enzyme showed significant release of xylose and a range of xylooligomers as shown in Table 14. This shows that Temer08570 has endo-xylanase with xylobiose, xylotriose and xylotetraose as main products.
- Table 14 Effect of Rasamsonia emersonii Temer08570 on release of xylose and xylose oligomers from several xylan substrates after incubation for 20h at pH 4.5 and 60 °C at a dosage of 10 mg/g DM.
- ug/mL Substrate (2 mg/mL) xylose xylobiose xylotriose xylotetraose Beech wood xylan 5 19 25 25 Birch wood xylan 4 14 17 17 Oat arabinoxylan 0 16 18 15 * All substrates contain ⁇ 3.5 ug/mL of each product measured if no enzyme is added
- the endo-xylanase activity of Rasamsonia emersonii Temer08163 was analysed as described above.
- the supernatant of the Temer08163 A. niger shake flask fermentation was concentrated and added to several xylan substrates at 10 mg/g followed by incubation for 20 hours at pH 4,5 and 60°C.
- the enzyme showed significant release of xylbiose and xylose as shown in Table 15. This shows that Temer08570 has endo-xylanase activity with xylobiose as main product which was 12 - 25 times higher than the amount of xylose released.
- Table 15 Effect of Rasamsonia emersonii Temer08163 on release of xylose and xylose oligomers from several xylan substrates after incubation for 20h at pH 4.5 and 60 °C at a dosage of 10 mg/g DM.
- the alpha-glucuronidase activity of Rasamsonia emersonii Temer07305 was analysed as described above.
- the supernatant of the Temer07305 A. niger shake flask fermentation was concentrated and added to aldouronic acids both 1 and 10 mg/g followed by incubation for 24 hours at pH 4,5 and 60°C.
- the enzyme was able to remove 4-O-methylglucuronic acid from the xyloilogomers resulting in the simultaneous release of xylose, xylobiose, xylotriose and xylotatraose as shown in Table 16. This shows that Temer07305 has alpha-glucuronidase activity.
- Table 16 The release of xylose and xylose oligomers by Rasamsonia emersonii Temer07305 from aldouronic acids as a result of the hydrolysis of 4-O-methylglucuronic acid from these xylooligomers, after incubation for 24h at pH 4.5 and 60 °C at a dosage of 1 and 10 mg/g DM.
- Area/ (mg/mL) substrate Protein ID Dosage (mg/g DM) xylose xylobiose xylotriose xylotetraose No enzyme x 25 6 0 0 Temer07305 1 45 180 58 19 Temer07305 10 120 180 55 9
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Claims (15)
- Polypeptide ayant une activité d'hémicellulase, qui comprend la séquence d'acides aminés indiquée dans la SEQ ID n° : 72 ou une séquence d'acides aminés codée par la séquence nucléotidique de SEQ ID n° : 71, SEQ ID n° : 74, ou bien polypeptide variant ou polynucléotide variant de celui-ci, le polypeptide variant ayant une identité de séquence d'au moins 75% avec la séquence indiquée dans la SEQ ID n° : 72 ou le polynucléotide variant codant pour un polypeptide qui a une identité de séquence d'au moins 75% avec la séquence indiquée dans la SEQ ID n° : 72.
- Polypeptide, selon la revendication 1, ayant une activité d'alpha-glucuronidase.
- Séquence d'acides nucléiques isolée codant pour une hémicellulase, la séquence d'acides nucléiques étant choisie dans le groupe constitué par :(a) une séquence d'acides nucléiques ayant une identité d'au moins 70% avec la séquence d'acides nucléiques de SEQ ID n° : 71, SEQ ID n° : 74 ou SEQ ID n° : 75 ;(b) une séquence d'acides nucléiques codant pour (i) la séquence d'acides aminés de SEQ ID n° : 72, (ii) une séquence d'acides aminés ayant une identité d'au moins 75% avec la séquence d'acides aminés de SEQ ID n° : 72 ou (iii) une séquence d'acides aminés qui diffère de par 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ou 12 acide (s) aminé(s) de la séquence d'acides aminés de SEQ ID n° : 72 ; ou(c) une séquence d'acides nucléiques qui est le complément inverse d'une séquence d'acides nucléiques telle que définie dans les paragraphes (a) ou (b).
- Construction d'acides nucléiques ou vecteur comprenant la séquence d'acides nucléiques selon la revendication 3.
- Cellule recombinante comprenant un polypeptide selon les revendications 1 ou 2, une séquence d'acides nucléiques, selon la revendication 3, ou bien une construction d'acides nucléiques ou un vecteur selon la revendication 4, de préférence la cellule étant une cellule de champignon, de préférence une cellule de champignon choisie dans le groupe constitué par les genres Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Rasamsonia, Thermoascus, Thielavia, Tolypocladium et Trichoderma.
- Cellule selon la revendication 5, dans laquelle un ou plusieurs gène(s) est/sont totalement ou en partie supprimé(s), inactivé(s) ou désorganisé(s), en option dans laquelle le gène code pour une protéase.
- Procédé destiné à la préparation d'un polypeptide, selon les revendications 1 ou 2, ayant une activité d'hémicellulase, lequel procédé comprend les étapes consistant à cultiver une cellule, selon les revendications 5 ou 6, dans des conditions qui permettent d'obtenir une expression dudit polypeptide et, en option, à récupérer le polypeptide exprimé.
- Composition comprenant : (i) un polypeptide selon les revendications 1 ou 2 et ; (ii) une cellulase et/ou une hémicellulase supplémentaire et/ou une pectinase.
- Composition selon la revendication 8, dans laquelle la cellulase est une GH61, cellobiohydrolase I, cellobiohydrolase II, endo-β-1,4-glucanase, β-glucosidase ou β-(1,3)(1,4)-glucanase et/ou dans laquelle l'hémicellulase supplémentaire est une endoxylanase, β-xylosidase, α-L-arabino-furanosidase, α-D-glucuronidase, féruloyl estérase, coumaroyl estérase, α-galactosidase, β-galactosidase, β-mannanase ou β-mannosidase.
- Composition selon les revendications 8 ou 9, la composition étant un bouillon brut de fermentation d'une masse cellulaire.
- Procédé destiné au traitement d'un substrat comprenant l'hémicellulose, en option un matériel végétal, lequel procédé comprend une mise en contact du substrat avec un polypeptide, selon les revendications 1 ou 2, et/ou une composition selon l'une quelconque des revendications 8 à 10.
- Procédé selon la revendication 11, dans lequel, avant un traitement enzymatique, le substrat est prétraité avec une modification thermique, mécanique et/ou chimique ou une quelconque combinaison de tels procédés.
- Procédé selon les revendications 11 ou 12, dans lequel le traitement enzymatique est réalisé à une température de 50°C ou plus.
- Procédé destiné à produire un produit de fermentation, lequel procédé comprend les étapes consistant à produire un sucre fermentescible par une mise en contact d'un matériel ligno-cellulosique avec un polypeptide, selon les revendications 1 ou 2, et/ou une composition, selon l'une quelconque des revendications 8 à 10, et à fermenter le sucre fermentescible résultant, en produisant de ce fait le produit de fermentation.
- Utilisation d'un polypeptide, selon les revendications 1 ou 2, et/ou d'une composition, selon l'une quelconque des revendications 8 à 10, pour produire un sucre à partir d'un matériel ligno-cellulosique.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP18193710.3A EP3447136B1 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide à dégradation de carbohydrate et ses utilisations |
DK18193710.3T DK3447136T3 (da) | 2013-02-04 | 2014-02-03 | Carbohydratdegraderende polypeptid og anvendelser deraf |
PL18193710T PL3447136T3 (pl) | 2013-02-04 | 2014-02-03 | Polipeptyd degradujący węglowodany i jego zastosowania |
EP20154581.1A EP3686280B1 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide à dégradation de carbohydrate et ses utilisations |
Applications Claiming Priority (33)
Application Number | Priority Date | Filing Date | Title |
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EP13153841 | 2013-02-04 | ||
EP13153823 | 2013-02-04 | ||
EP13153828 | 2013-02-04 | ||
EP13153839 | 2013-02-04 | ||
EP13153836 | 2013-02-04 | ||
EP13153824 | 2013-02-04 | ||
EP13153833 | 2013-02-04 | ||
EP13153831 | 2013-02-04 | ||
EP13153840 | 2013-02-04 | ||
EP13153834 | 2013-02-04 | ||
EP13153835 | 2013-02-04 | ||
EP13153829 | 2013-02-04 | ||
EP13153821 | 2013-02-04 | ||
EP13153837 | 2013-02-04 | ||
EP13153825 | 2013-02-04 | ||
EP13156693 | 2013-02-26 | ||
EP13156692 | 2013-02-26 | ||
EP13156684 | 2013-02-26 | ||
EP13156702 | 2013-02-26 | ||
EP13156701 | 2013-02-26 | ||
EP13156698 | 2013-02-26 | ||
EP13156685 | 2013-02-26 | ||
EP13156694 | 2013-02-26 | ||
EP13156696 | 2013-02-26 | ||
EP13156679 | 2013-02-26 | ||
EP13156682 | 2013-02-26 | ||
EP13156688 | 2013-02-26 | ||
EP13156690 | 2013-02-26 | ||
EP13156678 | 2013-02-26 | ||
EP13156703 | 2013-02-26 | ||
PCT/EP2014/051998 WO2014118360A2 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide dégradant les glucides et utilisations associées |
EP16158154.1A EP3070165A1 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide à dégradation de carbohydrate et ses utilisations |
EP14702266.9A EP2951296A2 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide dégradant les glucides et utilisations associées |
Related Parent Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP16158154.1A Division EP3070165A1 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide à dégradation de carbohydrate et ses utilisations |
EP16158154.1A Previously-Filed-Application EP3070165A1 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide à dégradation de carbohydrate et ses utilisations |
EP14702266.9A Division EP2951296A2 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide dégradant les glucides et utilisations associées |
Related Child Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP20154581.1A Division EP3686280B1 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide à dégradation de carbohydrate et ses utilisations |
EP18193710.3A Division EP3447136B1 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide à dégradation de carbohydrate et ses utilisations |
EP18193710.3A Division-Into EP3447136B1 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide à dégradation de carbohydrate et ses utilisations |
Publications (2)
Publication Number | Publication Date |
---|---|
EP3202900A1 EP3202900A1 (fr) | 2017-08-09 |
EP3202900B1 true EP3202900B1 (fr) | 2018-12-12 |
Family
ID=51263073
Family Applications (5)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP17156056.8A Active EP3202900B1 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide à dégradation de carbohydrate et ses utilisations |
EP16158154.1A Withdrawn EP3070165A1 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide à dégradation de carbohydrate et ses utilisations |
EP14702266.9A Withdrawn EP2951296A2 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide dégradant les glucides et utilisations associées |
EP18193710.3A Active EP3447136B1 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide à dégradation de carbohydrate et ses utilisations |
EP20154581.1A Active EP3686280B1 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide à dégradation de carbohydrate et ses utilisations |
Family Applications After (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP16158154.1A Withdrawn EP3070165A1 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide à dégradation de carbohydrate et ses utilisations |
EP14702266.9A Withdrawn EP2951296A2 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide dégradant les glucides et utilisations associées |
EP18193710.3A Active EP3447136B1 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide à dégradation de carbohydrate et ses utilisations |
EP20154581.1A Active EP3686280B1 (fr) | 2013-02-04 | 2014-02-03 | Polypeptide à dégradation de carbohydrate et ses utilisations |
Country Status (11)
Country | Link |
---|---|
US (4) | US9988615B2 (fr) |
EP (5) | EP3202900B1 (fr) |
KR (1) | KR102134498B1 (fr) |
CN (1) | CN105073986A (fr) |
CA (1) | CA2900140C (fr) |
DK (3) | DK3686280T3 (fr) |
ES (2) | ES2802807T3 (fr) |
HR (2) | HRP20220917T1 (fr) |
MY (2) | MY187542A (fr) |
PL (2) | PL3686280T3 (fr) |
WO (1) | WO2014118360A2 (fr) |
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US11447759B2 (en) | 2013-02-04 | 2022-09-20 | Dsm Ip Assets B.V. | Carbohydrate degrading polypeptide and uses thereof |
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US11499142B2 (en) | 2016-11-24 | 2022-11-15 | Dsm Ip Assets B.V. | Enzyme composition |
US20190276809A1 (en) | 2016-11-24 | 2019-09-12 | Dsm Ip Assets B.V. | Enzyme composition |
WO2018185071A1 (fr) | 2017-04-03 | 2018-10-11 | Dsm Ip Assets B.V. | Procédé pour l'hydrolyse enzymatique de matière lignocellulosique et la fermentation de sucres |
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EP3704259A1 (fr) | 2017-10-30 | 2020-09-09 | DSM IP Assets B.V. | Procédé pour l'hydrolyse enzymatique de matière lignocellulosique et la fermentation de sucres |
WO2019086370A1 (fr) | 2017-10-30 | 2019-05-09 | Dsm Ip Assets B.V. | Procédé pour l'hydrolyse enzymatique de matière lignocellulosique et la fermentation de sucres |
CN107988127B (zh) * | 2017-11-02 | 2021-09-03 | 南京农业大学 | 里氏木霉木质纤维素酶基因工程乳酸菌组合在调制优质苜蓿青贮饲料中的应用 |
WO2019185681A1 (fr) | 2018-03-28 | 2019-10-03 | Dsm Ip Assets B.V. | Composition enzymatique |
EP3775189A1 (fr) | 2018-03-28 | 2021-02-17 | DSM IP Assets B.V. | Composition enzymatique |
WO2019219804A1 (fr) | 2018-05-17 | 2019-11-21 | Dsm Ip Assets B.V. | Procédé de production d'un polypeptide |
HUE061611T2 (hu) | 2018-05-30 | 2023-07-28 | Versalis Spa | Eljárás cukrok elõállítására szénhidrát-anyagokból |
WO2020058248A1 (fr) | 2018-09-18 | 2020-03-26 | Dsm Ip Assets B.V. | Procédé d'hydrolyse enzymatique de matière glucidique et fermentation de sucres |
WO2020058249A1 (fr) | 2018-09-18 | 2020-03-26 | Dsm Ip Assets B.V. | Procédé pour l'hydrolyse enzymatique de matière glucidique et la fermentation de sucres |
WO2020058253A1 (fr) | 2018-09-18 | 2020-03-26 | Dsm Ip Assets B.V. | Procédé d'hydrolyse enzymatique de matière glucidique et fermentation de sucres |
BR112021006739A2 (pt) | 2018-10-24 | 2021-07-13 | Dsm Ip Assets B.V. | processo para hidrólise enzimática de material de carboidrato e fermentação de açúcares |
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BR112021017167A2 (pt) | 2019-03-12 | 2021-11-09 | Dsm Ip Assets Bv | Processo para a produção de um caldo de fermentação |
MX2022002436A (es) * | 2019-08-30 | 2022-06-02 | Ab Enzymes Gmbh | Uso de celulas gh12 en horneado de centeno. |
EP4028536A1 (fr) | 2019-09-10 | 2022-07-20 | DSM IP Assets B.V. | Composition enzymatique |
CN110577960B (zh) * | 2019-09-12 | 2022-09-13 | 南京农业大学 | 梨木质素合成基因PbMC1a/1b及其在果实品质遗传改良中的应用 |
CN110791439B (zh) * | 2019-10-10 | 2022-04-19 | 天津科技大学 | 一株基因工程构建发酵生产苹果酸的重组黑曲霉菌株及应用 |
US20230313257A1 (en) * | 2020-06-05 | 2023-10-05 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and Systems to Secrete Lignin-Modifying Enzymes and Uses Thereof |
WO2022013148A1 (fr) | 2020-07-13 | 2022-01-20 | Dsm Ip Assets B.V. | Procédé de production de biogaz |
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2014
- 2014-02-03 CN CN201480019746.5A patent/CN105073986A/zh active Pending
- 2014-02-03 WO PCT/EP2014/051998 patent/WO2014118360A2/fr active Application Filing
- 2014-02-03 PL PL20154581.1T patent/PL3686280T3/pl unknown
- 2014-02-03 ES ES18193710T patent/ES2802807T3/es active Active
- 2014-02-03 DK DK20154581.1T patent/DK3686280T3/da active
- 2014-02-03 EP EP17156056.8A patent/EP3202900B1/fr active Active
- 2014-02-03 EP EP16158154.1A patent/EP3070165A1/fr not_active Withdrawn
- 2014-02-03 DK DK17156056.8T patent/DK3202900T3/en active
- 2014-02-03 EP EP14702266.9A patent/EP2951296A2/fr not_active Withdrawn
- 2014-02-03 CA CA2900140A patent/CA2900140C/fr active Active
- 2014-02-03 MY MYPI2019001267A patent/MY187542A/en unknown
- 2014-02-03 EP EP18193710.3A patent/EP3447136B1/fr active Active
- 2014-02-03 EP EP20154581.1A patent/EP3686280B1/fr active Active
- 2014-02-03 HR HRP20220917TT patent/HRP20220917T1/hr unknown
- 2014-02-03 ES ES20154581T patent/ES2923460T3/es active Active
- 2014-02-03 DK DK18193710.3T patent/DK3447136T3/da active
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Publication number | Priority date | Publication date | Assignee | Title |
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US11447759B2 (en) | 2013-02-04 | 2022-09-20 | Dsm Ip Assets B.V. | Carbohydrate degrading polypeptide and uses thereof |
Also Published As
Publication number | Publication date |
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EP2951296A2 (fr) | 2015-12-09 |
EP3070165A1 (fr) | 2016-09-21 |
EP3686280B1 (fr) | 2022-06-22 |
PL3447136T3 (pl) | 2020-10-19 |
US20150361408A1 (en) | 2015-12-17 |
DK3202900T3 (en) | 2019-04-08 |
HRP20220917T1 (hr) | 2022-10-28 |
KR102134498B1 (ko) | 2020-07-16 |
WO2014118360A2 (fr) | 2014-08-07 |
MY176331A (en) | 2020-07-29 |
US10655115B2 (en) | 2020-05-19 |
CA2900140C (fr) | 2022-05-24 |
US20180245059A1 (en) | 2018-08-30 |
WO2014118360A3 (fr) | 2014-09-25 |
EP3447136A1 (fr) | 2019-02-27 |
DK3686280T3 (da) | 2022-09-12 |
US20190136213A1 (en) | 2019-05-09 |
CN105073986A (zh) | 2015-11-18 |
CA2900140A1 (fr) | 2014-08-07 |
US11447759B2 (en) | 2022-09-20 |
EP3202900A1 (fr) | 2017-08-09 |
PL3686280T3 (pl) | 2022-09-19 |
ES2923460T3 (es) | 2022-09-27 |
KR20150113170A (ko) | 2015-10-07 |
DK3447136T3 (da) | 2020-08-03 |
HRP20200976T1 (hr) | 2020-11-13 |
US10316305B2 (en) | 2019-06-11 |
EP3686280A1 (fr) | 2020-07-29 |
EP3447136B1 (fr) | 2020-05-13 |
US9988615B2 (en) | 2018-06-05 |
US20200190495A1 (en) | 2020-06-18 |
ES2802807T3 (es) | 2021-01-21 |
MY187542A (en) | 2021-09-28 |
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